Team:UNICAMP-Brazil/Notebooks/October 7

ColiGuard

Colony PCR and changing strategy

• A lot of colonies have grown with the reaction with only BBa B0015 and in the reaction with BBa B0015 + BBa K112806, we selected 15 to do a Colony PCR, none of them was positive. These results show that coudn't do a good digestion of BBa B0015, so tha's why it's recircularizing and giving a lot of fake positives.

• To improve our digestion we let BBa B0015 digesting ON with EcoRI and tomorrow we will purify it from agarose gel to make sure we only get linearized plasmid, then we will digest with XbaI tomorrow.

Marcos

YeastGuard

New biobricks - New strategy

- We performed PCR of 7 colonies of each construction (Jen1_orf and Jen1_promoter) inoculated yesterday. We found 4 positive colonies for Jen1_promoter! =)

GEL

- Unfortunately no Jen1_orf construction was found in but we continued screenig! To find positive colonies we screened 10 more colonies and made a backup inoculum in case we find right ones.

- We found no fragments again!! Maybe the ligation wasn't succesfull. We will repeat it tomorrow.

- We also made 10 colonies PCR for the following constructions: pGEM-Lisozyme and the biobrick YFP-end. We found 7 positive colonies for pGEM-Lisozyme =) Moreover we found some strange fragments in YFP-End PCR. This fragaments correspond to the vectors size (YFP Biobrick), without the insert (End). We believe that this fragment correspond to the recircularized vactor. Our expected fragment wouldn't be amplified, since it has 3000pb, so we decided to perform miniprep from the colonies that showed no amplicons! We hope we are right! =)

- We did the yest pre-inoculun to make competent cells.

Raíssa