Team:UNICAMP-Brazil/Notebooks/September 29

YeastGuard

Dephosphorylation - CIAP test

• Only a few colonies were found this morning =(. Unfortunately the number of colonies wasn´t enough to compare and decide between CIAP and SAP. So we decided to use SAP to dephosphorylate our biofusion vector, considering that the protocol is much easier.

New biobricks

• Today we performed 5' dephosphorylation of the biofusion vector with SAP (Protocol 9). We did the ligation reaction (Protocol 11) with the 5' dephosphorylated vector and the three digested (XbaI and SpeI) new parts (pJen1, pDLD and Lysozyme).

• We transformed the thermocompetent E. coli (Protocol 3) with the ligations and plated in LB+Amp media.

Raíssa and Taís

Customizing the PCR

• Gradient PCR of the Jen1 (ORF) using the melting temperatures: 53ºC, 55ºC and 57ºC. How you can see in the gel, this amplification wasn’t so good. Then the strategy will be: join the three products obtained by this gradient PCR and make a electrophoresis to purify the band corresponding to the Jen1 (ORF) size and ,then, make a new PCR.

• To get a optimal PCR that can amplify the …… using the primers VF and VR we did a gradient PCR. The temperatures tested were the same of the Jen1 (ORF) PCR.

• The electrophoresis didn’t show big differences into the three melting temperatures used and the bands pattern correspond to the size of ………

Wesley and Gleidson

ColiGuard

CeiB: Colony PCR

• <p We got transforming cells apparently. We made colony PCR with VF and VR primer in order to prove the transformation. The CeaB transformation was wrong, because of don’t show the right size band. We tried one more time transformation. Otherwise the CeiB transformation showed possible correct band size (~400), then to confirm, we are going to make miniprep tomorrow and PCR again.</p>

Luige

Miniprep Results

• We ran an agarose gel with the minipreps samples (proccedure performed yesterday).

• According to both pictures, we sucessfully recovered all biobricks! :)

Marcelo