Team:UNICAMP-Brazil/Notebooks/October 10

YeastGuard

pADH1+YFP (new biobrick)

• A electrophoresis was made with the product of the digestion performed yesterday. Then, we purify the bands corresponding to the size of the pADH1 in the biofusion vector and the YFP part.
• So we made the ligation of the YFP part in the biofusion vector that contains the ADH1 promoter. Then we transformed this construction in E. coli that growth over-night in the kanamicin media plate.
•  ??Digestão Lisozima (X e S)??

Wesley and Gleidson

ColiGuard

Transformation of the BBa K112806 + BBa B0015 ligation

• We purified the digestion from agarose gel and use the product to do a ligation with BBa B0014 + BBa K112806 according to Protocol 11.

• The ligation was used in the transformation of E. coli. We hope with BBa B0014 the digestion is better than B0015.

finO and finP with pGEM - confirmation

• Once we confirmed that several samples actually have finO and finP inserts ligated into pGEM vector (by the digestion procedure performed yesterday), we could proceed to the final step on confirming our finOP-pGEM ligations.

• We performed a PCR for each miniprep samples we got, using a specific forward primer for our inserts (finO and finP) and a reverse primer that is specific for pGEM vector (primer M13, see pGEM manual). This will allow us to verify that we have the insert ligated into the plasmid and, further more, will allow us to check whether or not our inserts are indeed in the correct frame position.

• According to the pictures, both finO and finP's PCR resulted in bands that reached the expected size in several samples.
• Therefore, we concluded we sucessfully have finO and finP correctly ligated into pGEM vector. Next step is ligating them into biobrick's vector.

Marcelo