http://www.lembiotech.ufc.br/wiki/index.php?action=history&feed=atom&title=DGGE_-_Passo_a_passo 2026-08-29T12:35:57Z Histórico de revisões para esta página nesta wiki MediaWiki 1.18.0 http://www.lembiotech.ufc.br/wiki/index.php?title=DGGE_-_Passo_a_passo&diff=444&oldid=prev 2011-09-05T18:31:36Z

<p></p> <table class='diff diff-contentalign-left'> <col class='diff-marker' /> <col class='diff-content' /> <col class='diff-marker' /> <col class='diff-content' /> <tr valign='top'> <td colspan='2' style="background-color: white; color:black;">← Versão anterior</td> <td colspan='2' style="background-color: white; color:black;">Edição de 18h31min de 5 de setembro de 2011</td> </tr><tr><td colspan="2" class="diff-lineno">Linha 455:</td> <td colspan="2" class="diff-lineno">Linha 455:</td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>:412 µL de água milli-Q</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>:412 µL de água milli-Q</div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>:Misturar tudo e aliquotar.</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>:Misturar tudo e aliquotar.</div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">==Observações e FAQ==</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">A bóia possui posição específica: o anel interno deve ficar para baixo; caso conntrário o equipamento não funcionará</ins></div></td></tr> </table>

Leon http://www.lembiotech.ufc.br/wiki/index.php?title=DGGE_-_Passo_a_passo&diff=443&oldid=prev 2011-09-05T18:29:22Z

<p><span class="autocomment">Protocolos</span></p> <table class='diff diff-contentalign-left'> <col class='diff-marker' /> <col class='diff-content' /> <col class='diff-marker' /> <col class='diff-content' /> <tr valign='top'> <td colspan='2' style="background-color: white; color:black;">← Versão anterior</td> <td colspan='2' style="background-color: white; color:black;">Edição de 18h29min de 5 de setembro de 2011</td> </tr><tr><td colspan="2" class="diff-lineno">Linha 297:</td> <td colspan="2" class="diff-lineno">Linha 297:</td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|+Programa colônia</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|+Programa colônia</div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|94°C||10 min</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|94°C||10 min</div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">|-</ins></div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|94°C||1 min||24X</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|94°C||1 min||24X</div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">|-</ins></div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|55°C||1min||24X</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|55°C||1min||24X</div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">|-</ins></div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|72°C||1 min||24X</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|72°C||1 min||24X</div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">|-</ins></div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|72°C||10 min</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|72°C||10 min</div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">|-</ins></div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|4°C </div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|4°C </div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|}</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|}</div></td></tr> <tr><td colspan="2" class="diff-lineno">Linha 402:</td> <td colspan="2" class="diff-lineno">Linha 407:</td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>#Incubar as placas a 4ºC para facilitar a seleção de colônias azuis das brancas.</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>#Incubar as placas a 4ºC para facilitar a seleção de colônias azuis das brancas.</div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"></td></tr> <tr><td class='diff-marker'>−</td><td style="background: #ffa; color:black; font-size: smaller;"><div>&#160;</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">===Meios de cultura===</ins></div></td></tr> <tr><td class='diff-marker'>−</td><td style="background: #ffa; color:black; font-size: smaller;"><div>&#160;</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>;Meio SOB (100 mL)</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>;Meio SOB (100 mL)</div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>*Triptona: 2 g</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>*Triptona: 2 g</div></td></tr> <tr><td colspan="2" class="diff-lineno">Linha 424:</td> <td colspan="2" class="diff-lineno">Linha 428:</td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>:;X-gal:</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>:;X-gal:</div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>::80 µg/mL (concentração final) - dissolvido em dimetilformamida, coberto com papel alumínio e estocado a 20 °C.</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>::80 µg/mL (concentração final) - dissolvido em dimetilformamida, coberto com papel alumínio e estocado a 20 °C.</div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">;Composição TBE (Tris-Borato-EDTA) 5X</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">:54 g Tris</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">:27,5 g de ácido bórico</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">:3,72 g EDTA</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">:Água q.s.p. 1L</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">:pH 8,0</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">;Composição TA (tampão de amostra) 6X</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">:Azul de bromofenol 0,25% p/v</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">:Glicerol 30%</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">:TE</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">:pH 8,0</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">:Para 10 mL: 3 mL de glicerol e 0,025 g de azul de bromofenol. Completa com TE até o volume de 10 mL.</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">;Composição TE (Tris-EDTA)</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">:Tris 10 mM</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">:EDTA 1 mM</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">:pH 7,8 - 8,0</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">:Para 100 mL: 0,121 g Tris (MM: 121,1) e 0,0326 g de EDTA (MM: 326). Adiciona água milli-Q até completar o volume para :100 mL.</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">;Preparo de marcador 1Kb DNA ladder (1 mL)</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">:75 µL de DNA padrão</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">:501 µL de tampão TA 6X</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">:12 µL de NaCl 5 M</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">:412 µL de água milli-Q</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">:Misturar tudo e aliquotar.</ins></div></td></tr> </table>

Leon http://www.lembiotech.ufc.br/wiki/index.php?title=DGGE_-_Passo_a_passo&diff=442&oldid=prev 2011-09-05T18:25:36Z

<p><span class="autocomment">Protocolos</span></p> <table class='diff diff-contentalign-left'> <col class='diff-marker' /> <col class='diff-content' /> <col class='diff-marker' /> <col class='diff-content' /> <tr valign='top'> <td colspan='2' style="background-color: white; color:black;">← Versão anterior</td> <td colspan='2' style="background-color: white; color:black;">Edição de 18h25min de 5 de setembro de 2011</td> </tr><tr><td colspan="2" class="diff-lineno">Linha 272:</td> <td colspan="2" class="diff-lineno">Linha 272:</td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>===Protocolo PCR de colônia===</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>===Protocolo PCR de colônia===</div></td></tr> <tr><td class='diff-marker'>−</td><td style="background: #ffa; color:black; font-size: smaller;"><div>{|</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>{|<ins class="diffchange diffchange-inline">width=&quot;500px&quot; align=&quot;center&quot;</ins></div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|+PCR de colônia</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|+PCR de colônia</div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|PCR Buffer (5X)||5</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|PCR Buffer (5X)||5</div></td></tr> <tr><td colspan="2" class="diff-lineno">Linha 294:</td> <td colspan="2" class="diff-lineno">Linha 294:</td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|Total||25ul</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|Total||25ul</div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|}</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|}</div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">{|width=&quot;500px&quot; align=&quot;center&quot;</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">|+Programa colônia</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">|94°C||10 min</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">|94°C||1 min||24X</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">|55°C||1min||24X</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">|72°C||1 min||24X</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">|72°C||10 min</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">|4°C </ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">|}</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">*Pega colônia com palito e coloca na água, depois acrescenta o mix</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">;Eletroforese fragmento 1500 pb</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">:0,8-1% agarose em TBE 0,5X</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">:Cubeta pequena: 25 mL</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">:Syber Safe: 0,5 µL</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">:100 V</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">;Eletroforese para cortar da banda do gel</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">:0,8-1% agarose em TBE 0,5X</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">:Cubeta grande: 90 mL</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">:Syber Safe: 1 µL</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">:Aplicar o volume de duas reações de PCR por poço (50 µL + 4 µL tampão de amostra)</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">:100V</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">===Purificação de DNA da banda do gel===</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">;Dissolvendo a banda do gel</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">#Após a eletroforese, excisar a banda do gel e colocar em um tubo de microcentrífuga de 1,5 mL (pesa o tubo antes e depois para saber o peso do gel)</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">#Adicionar 10 µL da solução Membrane Binding para cada 10 mg da banda do gel. Vortex e incubar a 50-65 °C até que dissolva completamente. (Max. 350 mg de gel ou 40 µg DNA/membrana).</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">;Ligação do DNA</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">#Inserir minicoluna SV dentro de tudo coletor.</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">#Transferir mistura de gel dissolvida. Incubar a temperatura ambiente por 1 minuto.</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">#Centrifugar a 16000 x g por 1 min (13400 rpm por 2 min). Descartar o sobrenadante e reinserir a minicoluna no tubo coletor.</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">;Lavagem</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">#Adicionar 700 µL da Solução Membrane Wash (adicionado de etanol). Centrifugar a 16000 g por 1 min (13400 rpm por 2 min). Descartar o sobrenadante e reinserir a minicoluna dentro do tubo coletor.</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">#Repetir o passo 4 com 500 µL de solução Membrane Wash. Centrifugar a 16000 g por 5 min (13400 rpm por 8 min).</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">#Esvaziar o tubo de coleta e recentrifugar a minicoluna + tubo coletor por 1 min com a tampa aberta para permitir a evaporação do etanol residual.</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">;Eluição</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">#Cuidadosamente transferir a minicoluna para um tubo de microcentrifuga 1,5 mL.</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">#Adicionar 50 µL (20 µL) de água livre de nuclease para minicoluna. Incubar a temperatura ambiente por 1 min. Centrifugar a 16000 g por 1 min (13400 rpm por 2 min).</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">#Descartar a minicoluna e estocar o DNA a 4 °C ou a -20 °C.</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">===Concentrar DNA===</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">O volume final do DNA eluído será de 100 µL. O DNA deve ser concentrado pela adição de 4 µL de NaCl 5M e invertido 3-5 vezes para misturar. Posteriormente adicionar 200 µL de etanol frio 100% e inverte 3-5 vezes para misturar. Centrifugar a 10000 g por 5 min a temperatura ambiente. Decanta todo o liquido. Remover o tanol residual em um dessecador, speed vácuo ou secar ao ar. Ressuspender o DNA precipitado em água estéril ou tris 10 mM estéril. </ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">Secar DNA em termomixer a 40°C por 3 hrs.</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">===Reação de ligação===</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">Calcular a quantidade de DNA na reação de ligação (3:1, 5:1, 8:1)</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">{ [qtde de vetor (ng) x tamanho do inserto (kb)]/tamanho do vetor (kb) } x { razão molar x [inserto/vetor]} = inserto (ng)</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">===Protocolo de ligação===</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">#Centrifugar o vetor e o DNA controle para coletar do fundo do tubo</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">#Usar tubos de 0,5 mL</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">#Agitar em vortex o tampão de ligação 2X antes de cada uso</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">##Preparar alíquotas do tampão</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">#Misturar a reação por pipetagem</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">#Incubar a reação por 1 hora a temperatura ambiente (24 °C) ou por 16 hs a 4 °C.</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">Obs: Se usar o tampão de ligação 10X acrescenta no tubo de ligação apenas 1 µL desse tampão, podendo completar o volume final com água ou mais DNA.</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">{|width=&quot;500px&quot; align=&quot;center&quot;</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">|+Tabela</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">|Reagente||Reação (µL)||Controle + (µL)||Controle background</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">|-</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">|Tampão de ligação 2X||5||5||5</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">|-</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">|Vetor p-Gem-Easy||1||1||1</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">|-</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">|Produto de PCR X||-||-||</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">|-</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">|DNA controle||-||2||-</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">|-</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">|T4 DNA ligase (30U/ µL)||1||1||1</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">|-</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">|Água q.s.p.||10||10||10</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">|}</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">===Transformação usando pGEM-T Easy vetor===</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">#Remover tubos da E. coli (50 µL) mantidas a -80 °C e colocar em um banho de gelo até descongelar (aproximadamente 5 min). Misturar as células batendo gentilmente no tubo.&#160; </ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">#Centrifugar os tubos contendo as reações de ligação para coletar o conteúdo do fundo do tubo. Adicionar 2 µL de cada reação de ligação para tubos contendo a E. coli. Gentilmente agite os tubos (devagar) para misturar a reação. </ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">===Protocolo de eletroporação===</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">;Antes de tudo, as cubetas devem estar previamente resfriadas (mantidas tb no gelo) até a eletroporação.</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">#Transferir conteúdo preparado anteriormente para cubetas de eletroporação</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">#De forma asséptica quando misturar as células e a ligação, coletar cuidadosamente e colocar no fundo da cubeta (4 e 2 mm)</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">#Levar a cubeta ao eletroporador, ajustar a voltagem a 2500V e dar um pulso, vai aparecer a voltagem real que foi alcançada.</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">#Dar outro pulso sem a cubeta para o eletroporador ficar pronto para uma nova amostra.</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">#Após eletroporação, adicionar rapidamente à cubeta de eletroporação 900 µL do meio SOC previamente aquecido a 37 °C. </ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">#Pipetar gentilmente de 3-4x, com pipeta de 1 mL para ressuspensão das células, e transferir para tubo Falcon de 15 mL estéril</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">#Deixar sob agitação a 150 rpm, 37 °C por 1,5 h.</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">Se usar uma cubeta de 1 mm usar voltagem de 1800V.</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">===Protocolo de quimiotransformação===</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">#Coloque o tubo contendo a mistura de células e ligação em gelo por 20 minutos e depois aqueça apor 45-50 segundos em banho-maria a 42ºC (sem agitação) e retorne os tubos imediatamente ao gelo por 2 minutos.</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">#Após a transformação, adicione 950 µl de meio SOC a temperatura ambiente aos tubos contendo as células transformadas.</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">#Incube por 1,5 h a 37ºC a 150 rpm.</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">===Protocolo de plaqueamento dos clones===</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">#Após a transformação plaquei 100 µl da cada cultura transformada em duplicata em placas com antibiótico. Se alto nº de colônias é requerido, as células podem ser centrifugadas 1000g/10min, ressuspendida em 200 µL de meio SOC e plaquei 100 µl em cada placa </ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">#Incube as placas overnight (16-24h) a 37ºC (± 100 colônias/placa)</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">#Incubar as placas a 4ºC para facilitar a seleção de colônias azuis das brancas.</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">;Meio SOB (100 mL)</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">*Triptona: 2 g</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">*Extrato de levedura: 0,5 g</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">*NaCl: 0,05 g</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">#Adicionar parte da água e agitar até dissolver</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">#Adicionar 1 mL de KCl 250 mM</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">#Ajustar o pH para 7,0 com NaOH 5N</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">#Ajustar o volume para 100 mL</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">#Autoclavar e quando esfriar adicionar 0,5 mL de MgCl2 2 M ou 1mL de MgCl2 1M.</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">;Meio SOC (10 mL)</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">*Adicionar 180 µL de glicose 20% estéril</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">;IPTG: 0,5 mM (concentração final)</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">:;Estoque 0,1 M:</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">::1,2 g de IPTG em 50 mL de água milli-Q. Filtrar e estocar a 4 °C</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">:;Ampicilina:</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">::100 µg/mL (concentração final) - dissolvido em água milli-Q, coberto com papel alumínio e estocado a 20 °C</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">:;X-gal:</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">::80 µg/mL (concentração final) - dissolvido em dimetilformamida, coberto com papel alumínio e estocado a 20 °C.</ins></div></td></tr> </table>

Leon http://www.lembiotech.ufc.br/wiki/index.php?title=DGGE_-_Passo_a_passo&diff=441&oldid=prev 2011-09-05T17:55:53Z

<p><span class="autocomment">Protocolos</span></p> <table class='diff diff-contentalign-left'> <col class='diff-marker' /> <col class='diff-content' /> <col class='diff-marker' /> <col class='diff-content' /> <tr valign='top'> <td colspan='2' style="background-color: white; color:black;">← Versão anterior</td> <td colspan='2' style="background-color: white; color:black;">Edição de 17h55min de 5 de setembro de 2011</td> </tr><tr><td colspan="2" class="diff-lineno">Linha 193:</td> <td colspan="2" class="diff-lineno">Linha 193:</td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>==Protocolos==</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>==Protocolos==</div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">===Protocolo PCR (rRNA 16S Bacteria)===</ins></div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>{|width=&quot;500px&quot; align=&quot;center&quot;</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>{|width=&quot;500px&quot; align=&quot;center&quot;</div></td></tr> <tr><td class='diff-marker'>−</td><td style="background: #ffa; color:black; font-size: smaller;"><div>|+<del class="diffchange diffchange-inline">Protocolo </del>PCR (rRNA 16S Bacteria)</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>|+PCR (rRNA 16S Bacteria)</div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|Concentração final||1 reação (µL)</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|Concentração final||1 reação (µL)</div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|-</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|-</div></td></tr> <tr><td colspan="2" class="diff-lineno">Linha 230:</td> <td colspan="2" class="diff-lineno">Linha 231:</td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|}</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|}</div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"></td></tr> <tr><td class='diff-marker'>−</td><td style="background: #ffa; color:black; font-size: smaller;"><div>&#160;</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">===Protocolo PCR (rRNA 16S Achaea)===</ins></div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>{|width=&quot;500px&quot; align=&quot;center&quot;</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>{|width=&quot;500px&quot; align=&quot;center&quot;</div></td></tr> <tr><td class='diff-marker'>−</td><td style="background: #ffa; color:black; font-size: smaller;"><div>|+<del class="diffchange diffchange-inline">Protocolo </del>PCR (rRNA 16S Achaea)</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>|+PCR (rRNA 16S Achaea)</div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|Concentração final||1 reação (µL)</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|Concentração final||1 reação (µL)</div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|-</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|-</div></td></tr> <tr><td colspan="2" class="diff-lineno">Linha 268:</td> <td colspan="2" class="diff-lineno">Linha 269:</td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|-</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|-</div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|4°C||</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|4°C||</div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">|}</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">===Protocolo PCR de colônia===</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">{|</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">|+PCR de colônia</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">|PCR Buffer (5X)||5</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">|-</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">|MgCl2 (25mM)||2</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">|-</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">|dNTPs (10 mM)||0,5</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">|-</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">|Primer M13F (5 µM)||2,5</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">|-</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">|Primer M13R (5 µM)||2,5</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">|-</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">|GoTaq (5U/µL)||0,2</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">|-</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">|Gelatina 1%||0,025</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">|-</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">|Total mix por reação||12,7</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">|-</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">|Água mais colônia||12,3</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">|-</ins></div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">|Total||25ul</ins></div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|}</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|}</div></td></tr> </table>

Leon http://www.lembiotech.ufc.br/wiki/index.php?title=DGGE_-_Passo_a_passo&diff=427&oldid=prev 2011-09-05T17:37:33Z

<p><span class="autocomment">Protocolos</span></p> <table class='diff diff-contentalign-left'> <col class='diff-marker' /> <col class='diff-content' /> <col class='diff-marker' /> <col class='diff-content' /> <tr valign='top'> <td colspan='2' style="background-color: white; color:black;">← Versão anterior</td> <td colspan='2' style="background-color: white; color:black;">Edição de 17h37min de 5 de setembro de 2011</td> </tr><tr><td colspan="2" class="diff-lineno">Linha 193:</td> <td colspan="2" class="diff-lineno">Linha 193:</td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>==Protocolos==</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>==Protocolos==</div></td></tr> <tr><td class='diff-marker'>−</td><td style="background: #ffa; color:black; font-size: smaller;"><div>{|</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>{|<ins class="diffchange diffchange-inline">width=&quot;500px&quot; align=&quot;center&quot;</ins></div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|+Protocolo PCR (rRNA 16S Bacteria)</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|+Protocolo PCR (rRNA 16S Bacteria)</div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|Concentração final||1 reação (µL)</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|Concentração final||1 reação (µL)</div></td></tr> <tr><td colspan="2" class="diff-lineno">Linha 215:</td> <td colspan="2" class="diff-lineno">Linha 215:</td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|Total||25 µL</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|Total||25 µL</div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|}</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|}</div></td></tr> <tr><td class='diff-marker'>−</td><td style="background: #ffa; color:black; font-size: smaller;"><div>{|width=&quot;500px&quot;</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>{|width=&quot;500px<ins class="diffchange diffchange-inline">&quot; align=&quot;center</ins>&quot;</div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|+Programa Bacteria</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|+Programa Bacteria</div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|94°C||5 min</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|94°C||5 min</div></td></tr> </table>

Leon http://www.lembiotech.ufc.br/wiki/index.php?title=DGGE_-_Passo_a_passo&diff=425&oldid=prev 2011-09-05T17:36:27Z

<p><span class="autocomment">Protocolos</span></p> <table class='diff diff-contentalign-left'> <col class='diff-marker' /> <col class='diff-content' /> <col class='diff-marker' /> <col class='diff-content' /> <tr valign='top'> <td colspan='2' style="background-color: white; color:black;">← Versão anterior</td> <td colspan='2' style="background-color: white; color:black;">Edição de 17h36min de 5 de setembro de 2011</td> </tr><tr><td colspan="2" class="diff-lineno">Linha 193:</td> <td colspan="2" class="diff-lineno">Linha 193:</td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>==Protocolos==</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>==Protocolos==</div></td></tr> <tr><td class='diff-marker'>−</td><td style="background: #ffa; color:black; font-size: smaller;"><div>{|<del class="diffchange diffchange-inline">width=&quot;500px&quot;</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>{|</div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|+Protocolo PCR (rRNA 16S Bacteria)</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|+Protocolo PCR (rRNA 16S Bacteria)</div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|Concentração final||1 reação (µL)</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|Concentração final||1 reação (µL)</div></td></tr> </table>

Leon http://www.lembiotech.ufc.br/wiki/index.php?title=DGGE_-_Passo_a_passo&diff=424&oldid=prev 2011-09-05T17:35:52Z

<p><span class="autocomment">Protocolos</span></p> <table class='diff diff-contentalign-left'> <col class='diff-marker' /> <col class='diff-content' /> <col class='diff-marker' /> <col class='diff-content' /> <tr valign='top'> <td colspan='2' style="background-color: white; color:black;">← Versão anterior</td> <td colspan='2' style="background-color: white; color:black;">Edição de 17h35min de 5 de setembro de 2011</td> </tr><tr><td colspan="2" class="diff-lineno">Linha 193:</td> <td colspan="2" class="diff-lineno">Linha 193:</td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>==Protocolos==</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>==Protocolos==</div></td></tr> <tr><td class='diff-marker'>−</td><td style="background: #ffa; color:black; font-size: smaller;"><div>{|width=&quot;500px<del class="diffchange diffchange-inline">&quot; align=&quot;center&quot; cellpadding=&quot;20&quot; cellspacing=&quot;0</del>&quot;</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>{|width=&quot;500px&quot;</div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|+Protocolo PCR (rRNA 16S Bacteria)</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|+Protocolo PCR (rRNA 16S Bacteria)</div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|Concentração final||1 reação (µL)</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|Concentração final||1 reação (µL)</div></td></tr> <tr><td colspan="2" class="diff-lineno">Linha 215:</td> <td colspan="2" class="diff-lineno">Linha 215:</td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|Total||25 µL</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|Total||25 µL</div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|}</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|}</div></td></tr> <tr><td class='diff-marker'>−</td><td style="background: #ffa; color:black; font-size: smaller;"><div>{|width=&quot;500px<del class="diffchange diffchange-inline">&quot; align=&quot;center&quot; cellpadding=&quot;20&quot; cellspacing=&quot;0</del>&quot;</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>{|width=&quot;500px&quot;</div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|+Programa Bacteria</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|+Programa Bacteria</div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|94°C||5 min</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|94°C||5 min</div></td></tr> </table>

Leon http://www.lembiotech.ufc.br/wiki/index.php?title=DGGE_-_Passo_a_passo&diff=421&oldid=prev 2011-09-05T17:32:54Z

<p><span class="autocomment">Protocolos</span></p> <table class='diff diff-contentalign-left'> <col class='diff-marker' /> <col class='diff-content' /> <col class='diff-marker' /> <col class='diff-content' /> <tr valign='top'> <td colspan='2' style="background-color: white; color:black;">← Versão anterior</td> <td colspan='2' style="background-color: white; color:black;">Edição de 17h32min de 5 de setembro de 2011</td> </tr><tr><td colspan="2" class="diff-lineno">Linha 193:</td> <td colspan="2" class="diff-lineno">Linha 193:</td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>==Protocolos==</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>==Protocolos==</div></td></tr> <tr><td class='diff-marker'>−</td><td style="background: #ffa; color:black; font-size: smaller;"><div>{|width=&quot;500px&quot; align=&quot;center&quot;</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>{|width=&quot;500px&quot; align=&quot;center<ins class="diffchange diffchange-inline">&quot; cellpadding=&quot;20&quot; cellspacing=&quot;0</ins>&quot;</div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|+Protocolo PCR (rRNA 16S Bacteria)</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|+Protocolo PCR (rRNA 16S Bacteria)</div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|Concentração final||1 reação (µL)</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|Concentração final||1 reação (µL)</div></td></tr> <tr><td colspan="2" class="diff-lineno">Linha 215:</td> <td colspan="2" class="diff-lineno">Linha 215:</td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|Total||25 µL</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|Total||25 µL</div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|}</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|}</div></td></tr> <tr><td class='diff-marker'>−</td><td style="background: #ffa; color:black; font-size: smaller;"><div>{|width=&quot;500px&quot; align=&quot;center&quot;</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>{|width=&quot;500px&quot; align=&quot;center<ins class="diffchange diffchange-inline">&quot; cellpadding=&quot;20&quot; cellspacing=&quot;0</ins>&quot;</div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|+Programa Bacteria</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|+Programa Bacteria</div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|94°C||5 min</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|94°C||5 min</div></td></tr> </table>

Leon http://www.lembiotech.ufc.br/wiki/index.php?title=DGGE_-_Passo_a_passo&diff=410&oldid=prev 2011-09-05T17:21:10Z

<p><span class="autocomment">Protocolos</span></p> <table class='diff diff-contentalign-left'> <col class='diff-marker' /> <col class='diff-content' /> <col class='diff-marker' /> <col class='diff-content' /> <tr valign='top'> <td colspan='2' style="background-color: white; color:black;">← Versão anterior</td> <td colspan='2' style="background-color: white; color:black;">Edição de 17h21min de 5 de setembro de 2011</td> </tr><tr><td colspan="2" class="diff-lineno">Linha 217:</td> <td colspan="2" class="diff-lineno">Linha 217:</td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>{|width=&quot;500px&quot; align=&quot;center&quot;</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>{|width=&quot;500px&quot; align=&quot;center&quot;</div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|+Programa Bacteria</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|+Programa Bacteria</div></td></tr> <tr><td class='diff-marker'>−</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">|</del></div></td><td colspan="2">&#160;</td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|94°C||5 min</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|94°C||5 min</div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|-</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|-</div></td></tr> </table>

Leon http://www.lembiotech.ufc.br/wiki/index.php?title=DGGE_-_Passo_a_passo&diff=409&oldid=prev 2011-09-05T17:20:25Z

<p><span class="autocomment">Protocolos</span></p> <table class='diff diff-contentalign-left'> <col class='diff-marker' /> <col class='diff-content' /> <col class='diff-marker' /> <col class='diff-content' /> <tr valign='top'> <td colspan='2' style="background-color: white; color:black;">← Versão anterior</td> <td colspan='2' style="background-color: white; color:black;">Edição de 17h20min de 5 de setembro de 2011</td> </tr><tr><td colspan="2" class="diff-lineno">Linha 193:</td> <td colspan="2" class="diff-lineno">Linha 193:</td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>==Protocolos==</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>==Protocolos==</div></td></tr> <tr><td class='diff-marker'>−</td><td style="background: #ffa; color:black; font-size: smaller;"><div>{|</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>{|<ins class="diffchange diffchange-inline">width=&quot;500px&quot; align=&quot;center&quot;</ins></div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|+Protocolo PCR (rRNA 16S Bacteria)</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|+Protocolo PCR (rRNA 16S Bacteria)</div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|Concentração final||1 reação (µL)</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|Concentração final||1 reação (µL)</div></td></tr> <tr><td colspan="2" class="diff-lineno">Linha 215:</td> <td colspan="2" class="diff-lineno">Linha 215:</td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|Total||25 µL</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|Total||25 µL</div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|}</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|}</div></td></tr> <tr><td class='diff-marker'>−</td><td style="background: #ffa; color:black; font-size: smaller;"><div>{|</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>{|<ins class="diffchange diffchange-inline">width=&quot;500px&quot; align=&quot;center&quot;</ins></div></td></tr> <tr><td class='diff-marker'>−</td><td style="background: #ffa; color:black; font-size: smaller;"><div>|+Programa</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>|+Programa <ins class="diffchange diffchange-inline">Bacteria</ins></div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|</div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|94°C||5 min</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|94°C||5 min</div></td></tr> <tr><td colspan="2" class="diff-lineno">Linha 232:</td> <td colspan="2" class="diff-lineno">Linha 232:</td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"></td></tr> <tr><td class='diff-marker'>−</td><td style="background: #ffa; color:black; font-size: smaller;"><div>{|</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>{|<ins class="diffchange diffchange-inline">width=&quot;500px&quot; align=&quot;center&quot;</ins></div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|+Protocolo PCR (rRNA 16S Achaea)</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|+Protocolo PCR (rRNA 16S Achaea)</div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|Concentração final||1 reação (µL)</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|Concentração final||1 reação (µL)</div></td></tr> <tr><td colspan="2" class="diff-lineno">Linha 256:</td> <td colspan="2" class="diff-lineno">Linha 256:</td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|Total||25 µL</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|Total||25 µL</div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|}</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|}</div></td></tr> <tr><td class='diff-marker'>−</td><td style="background: #ffa; color:black; font-size: smaller;"><div>{|</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>{|<ins class="diffchange diffchange-inline">width=&quot;500px&quot; align=&quot;center&quot;</ins></div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|+Programa Archaea</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|+Programa Archaea</div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|94°C||5 min</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|94°C||5 min</div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">|-</ins></div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|94°C||0,5 min||30X</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|94°C||0,5 min||30X</div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">|-</ins></div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|55°C||1min||30X</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|55°C||1min||30X</div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">|-</ins></div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|72°C||1 min||30X</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|72°C||1 min||30X</div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">|-</ins></div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|72°C||10 min</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|72°C||10 min</div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">|-</ins></div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|4°C||</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|4°C||</div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|}</div></td><td class='diff-marker'>&#160;</td><td style="background: #eee; color:black; font-size: smaller;"><div>|}</div></td></tr> </table>

Leon