Team:UNICAMP-Brazil/Notebooks/October 9
From 2009.igem.org
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* We decided that we are going to confirm such ligations by performing: | * We decided that we are going to confirm such ligations by performing: | ||
- | -A Digestion: with | + | -A Digestion: with ''Xba''I and ''Spe''I restriction enzymes, in order to release our parts from pGEM plasmid; |
-A PCR: performed with the specific forward primer for our insert and with the reverse primer for pGEM plasmid (M13), in order to confirm that, once our inserts are indeed in there, they are also in the correct frame position. | -A PCR: performed with the specific forward primer for our insert and with the reverse primer for pGEM plasmid (M13), in order to confirm that, once our inserts are indeed in there, they are also in the correct frame position. | ||
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''Fabi and Léo'' | ''Fabi and Léo'' | ||
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+ | ====Cre-Recombinase - Confirmation: Stage I==== | ||
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+ | *<p style=”text-align:justify;”>After performing minipreps (on October 5th) for 20 inoculated cultures, possibly harbouring our Cre-Recombinase without ATG's Biobricks, it's time to start the confirmation procudures. Regarding "confirmation procudures", we mean a two stages procedure: (1) perform a specific PCR for Cre-Recombinase and (2) perform a digestion, aiming in excising a fragment of expectable size from the pSB1A3 vector.</p> | ||
+ | *<p style=”text-align:justify;”>Today we did stage I, a PCR reaction using Cre's specific designed primers, running an agarose gel straight after reaction ended.</p> | ||
+ | *<p style=”text-align:justify;”>Thereby, according to Stage I.... we did it! Several samples appears to contain our Cre's Biobrick!</p> | ||
+ | *<p style=”text-align:justify;”>If we succeed in Stage II, we will finally be able to say we got a biobrick! =]</p> | ||
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+ | ''Víctor'' | ||
==''' YeastGuard '''== | ==''' YeastGuard '''== | ||
- | ====New | + | ====[https://2009.igem.org/Team:UNICAMP-Brazil/Protocols/pGEMStrategy New Strategy: pGEM]==== |
- | *<p style= | + | *<p style="text-align:justify;">The plates containing ''E. coli'' transformed with pDLD didn’t grow. We repeated the ligation reaction and transformed competent ''E. coli'' again.</p> |
*<p style=”text-align:justify;”>We transformed the ligation between Lysozyme and biofusion, performed yesterday, in competent ''E. coli'' and plated in LB+Amp media.</p> | *<p style=”text-align:justify;”>We transformed the ligation between Lysozyme and biofusion, performed yesterday, in competent ''E. coli'' and plated in LB+Amp media.</p> | ||
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====YFP+Terminator==== | ====YFP+Terminator==== | ||
*<p style=”text-align:justify;”>We transformed the ligation between YFP+end and Adh1 promoter, performed yesterday, in competent ''E. coli'' and plated in LB+Amp media.</p> | *<p style=”text-align:justify;”>We transformed the ligation between YFP+end and Adh1 promoter, performed yesterday, in competent ''E. coli'' and plated in LB+Amp media.</p> | ||
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+ | * We decided to check over the size of the terminator part at the registry, since we had to much work with it. We submited its sequence to the BLAST algorithm and discovered that this biobrick is the same as YFP one!!! =( We got so desapointed =/ It was the only terminator biobrick to be used in yeasts. | ||
====pADH1+YFP==== | ====pADH1+YFP==== | ||
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*<p style=”text-align:justify;”>First of all, we did these digestions: 1)YFP digestion using the enzymes ''Xba''I and ''Pst''I; 2)pADH1 (biofusion) digestion using the enzymes ''Spe''I and ''Pst''I.</p> | *<p style=”text-align:justify;”>First of all, we did these digestions: 1)YFP digestion using the enzymes ''Xba''I and ''Pst''I; 2)pADH1 (biofusion) digestion using the enzymes ''Spe''I and ''Pst''I.</p> | ||
- | ''Wesley | + | ''Wesley'' |
- | ==== | + | ====YEP358==== |
We transformed ''E. coli'' with YEP358-β galactosidase plasmid ([https://2009.igem.org/Team:UNICAMP-Brazil/Protocols/Electroporation Protocol 3]) | We transformed ''E. coli'' with YEP358-β galactosidase plasmid ([https://2009.igem.org/Team:UNICAMP-Brazil/Protocols/Electroporation Protocol 3]) | ||
{{:Team:UNICAMP-Brazil/inc_rodape}} | {{:Team:UNICAMP-Brazil/inc_rodape}} |
Latest revision as of 03:20, 22 October 2009
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