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Team:UNICAMP-Brazil/Notebooks/October 3
From 2009.igem.org
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====Ligation of finOP and Cre-Recombinase on pGEM vector==== | ====Ligation of finOP and Cre-Recombinase on pGEM vector==== | ||
| - | * In order to start our pGEM strategy on assembling biobricks, today we ligated PCR's products of finO, finP and Cre-Recombinase into pGEM Vector. There is no need for any digestion before this procedure, since pGEM has an T stick ends capable of pairing with the A stick ends left by Taq Polymerase on the PCR products. | + | *<p style=”text-align:justify;”>In order to start our pGEM strategy on assembling biobricks, today we ligated PCR's products of finO, finP and Cre-Recombinase into pGEM Vector. There is no need for any digestion before this procedure, since pGEM has an T stick ends capable of pairing with the A stick ends left by Taq Polymerase on the PCR products.</p> |
| - | * We followed [https://2009.igem.org/Team:UNICAMP-Brazil/Protocols/T4_DNA_Ligase Protocol 11], without modifications. | + | *<p style=”text-align:justify;”>We followed [https://2009.igem.org/Team:UNICAMP-Brazil/Protocols/T4_DNA_Ligase Protocol 11], without modifications.</p> |
| - | * Ligation lasted 1 hour. | + | *<p style=”text-align:justify;”>Ligation lasted 1 hour.</p> |
''Marcelo and Victor'' | ''Marcelo and Victor'' | ||
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====Transformation of finOP's and Cre-Recombinase's ligations==== | ====Transformation of finOP's and Cre-Recombinase's ligations==== | ||
| - | * After performing the required ligations, we then transformed them into electrocompetent E. coli bacteria, strain DH10B, according to [https://2009.igem.org/Team:UNICAMP-Brazil/Protocols/Electroporation Protocol 3]. | + | *<p style=”text-align:justify;”>After performing the required ligations, we then transformed them into electrocompetent E. coli bacteria, strain DH10B, according to [https://2009.igem.org/Team:UNICAMP-Brazil/Protocols/Electroporation Protocol 3].</p> |
| - | * We plated the trasformed cells in LB-AMP media, which already contained X-gal substrate. | + | *<p style=”text-align:justify;”>We plated the trasformed cells in LB-AMP media, which already contained X-gal substrate.</p> |
| - | * Plates were incubated at 37ºC for an O/N period. | + | *<p style=”text-align:justify;”>Plates were incubated at 37ºC for an O/N period. </p> |
''Marcelo and Victor'' | ''Marcelo and Victor'' | ||
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====New biobricks - New strategy (pGEM)==== | ====New biobricks - New strategy (pGEM)==== | ||
| - | * The ligation reactions didn´t work. So today we decided not to digest the fragment and the pGEM vector with ''SpeI'', but to ligate it directly, considering that we can confirm the correct insertion by PCR. | + | *<p style=”text-align:justify;”>The ligation reactions didn´t work. So today we decided not to digest the fragment and the pGEM vector with ''SpeI'', but to ligate it directly, considering that we can confirm the correct insertion by PCR.</p> |
| - | * We did new ligation reactions with our four parts (pJEN1, pDLD, Lysozyme and orfJEN1) in pGEM. | + | *<p style=”text-align:justify;”>We did new ligation reactions with our four parts (pJEN1, pDLD, Lysozyme and orfJEN1) in pGEM.</p> |
''Taís'' | ''Taís'' | ||
{{:Team:UNICAMP-Brazil/inc_rodape}} | {{:Team:UNICAMP-Brazil/inc_rodape}} | ||
Revision as of 18:10, 20 October 2009
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