Team:UNICAMP-Brazil/Notebooks/September 23

ColiGuard

F plasmid recircularization

• The ligation reaction was assembled according to Protocol 11. Both the plasmid samples were used to recircularization. The reaction tubes was kept O/N at 4°C.

Gabriel

finO and finP - Still Trying to Confirm our Biobricks

• Today we performed minipreps from yesterday's inoculated cultures, according to Protocol 2.

Marcelo

PY Promoter - New strategy

• Our cloning strategy for inserting PY1 and PY2 fragments into the plasmid containing the RFP reporter didn’t work as we expected. We believe that one of our problems is the compatible cohesive ends produced by the enzymes XbaI and SpeI. As other members of our team are facing this same problem, our group decided to create another strategy to construct our biobricks. This new strategy is based on the pGEM vector system and consists basically in cloning our fragments in this vector and then excising them with EcoRI and SpeI. After the excision our fragment won’t have compatible cohesive ends anymore. (See pGEM cloning strategy for more information).

• So today we started to work in this new strategy. First of all we digested our fragments PY1 and PY2 (amplified by PCR from F plasmid) and pGEM vector with SpeI. The digestion lasted for 3 hours.

• After the digestion we performed 2 ligation reactions following Protocol 11: PY1 + pGEM and PY2 + pGEM.

Fabi and Léo

CeaB and CeiB: colony PCR

• We got apparently good transformations but we have to prove the correct position of DNA inserted. So, we performed colony PCR with right primers.

We didn’t get the band size expected and we found unspecific amplification, therefore we will make miniprep and we will try PCR again.

Ane