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ColiGuard
CRE-Recombinase without ATG - Miniprep
Victor
finOP and Cre-Recombinase with pGEM strategy
- Today we performed minipreps from yesterday's inoculated cultures, according to Protocol 2.
- We expect to recover pGEM plasmids finally containing singly our inserts for finO, finP and Cre-Recombinase w/o ATG.
- As both sides of the inserts contains the A stick end, it's possible that we will find in some samples unwanted ligations (it could be ligate in an upside down position). Therefore, we will also need to perform PCRs to confirm that our inserts indeed are in the correct frame position.
Marcelo and Victor
Colony PCR to confirm the BBa K112806 + BBa B0015 ligation
A lot of colonies have grown, it looks like our ligation were ok.
We selected 10 colonies to a PCR colonies,we used BBa B0015 as our positive control.
All the colonies didn't have the insertion of BBa K112806, but the size of the fragment was ok to BBa B0015.
We don't have material enough of BBa B0015 for other digestion, so we did another inoculum to do a miniprep tomorrow.
Marcos, Luige and Ane
PY Promoter - Colony-PCR screening
Unfortunately, just the colonies with PY1 displayed bands with the expected size in the gel. Since the sequence of PY1 is more complete than the sequence of PY2, we decided to not try again with PY2 and use just the colonies that have pGEM vector with PY1.
Fabi and Léo
CeaB and CeiB: pGEM cloning strategy
- Due to the unsuccessful transformations in the backbone plasmid, we decided to change the strategy. Then, today we started the pGem cloning strategy (Protocol 15). We are going to insert our DNA colicins in the pGEM plasmid. We cut CeaB, CeiB and pGEM plasmid with SpeI restriction enzyme separately. After purification, we started the ligation reaction. We joined pGEM plasmid with CeaB and CeiB respectively. We made dyalisis and finally transformed into competent E.coli. We expect a good result this time.
Ane and Luige
YeastGuard
Raíssa and Taís
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