Team:UNICAMP-Brazil/Notebooks/October 9

ColiGuard

Another PCR colony of BBa K112806 + BBa B0015, another failure...

• A lot of colonies have grown again, we selected 15 to do the Colony PCR, but none of them was positive.

• We are really worried with this, our time is finishing. We decided to chenge radically our strategy. We will change the terminator to BBa B0014 instead of BBa B0015 because the digestion of B0015 is not working. We let B0014 digesting ON with EcoRI and XbaI

Marcos

finO and finP with pGEM - confirmation

• We decided that we are going to confirm such ligations by performing:

-A Digestion: with XbaI and SpeI 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.

• Seeing that, today we performed the digestion with EcoRI and SpeI for all of our minipreps samples that actually worked. Digestion lasted 3 hours.

• According to the picture, the digestion excised a fragment of expected size (600 bp for finO and 120 bp for finP) in almost all samples! That's an important clue on confirming our inserts's correctly ligation!

Gabriel and Marcelo

YeastGuard

pADH1+YFP (new biobrick)

• Today we start the experiments to obtain a new biobrick. It’s the ADH1+YFP biobrick.
• First of all, we did these digestions:1)YFP digestion using the enzymes XbaI and PstI; 2)pADH1 (bioffusion) digestion using the enzymes SpeI and PstI

Wesley and Gleidson