Team:UNICAMP-Brazil/Notebooks/October 5

ColiGuard

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 innoculum to do a miniprep tomorrow

Marcos, Luige and Ane

YeastGuard

New strategy: pGEM

• We digested the biofusion vector with two combinations of enzymes: EcoRI and SpeI; XbaI and PstI to use in the new strategy (Protocol X).

GEL

• We repeated the part's PCR to be sure that we will have enough material to perform the following ligation reactions to achieve the biobrick format. We obtained orfJEN1, pJEN1, pDLD and Lysozyme amplicons.

GEL

• We confirmed the correct insertion of pDLD in pGEM by colony PCR using the part's internal forward primer and the vector's reverse primer (M13). The Lysozyme insertion wasn’t confirmed yet.

GEL