Team:UNICAMP-Brazil/Notebooks/September 25

ColiGuard

Getting the visa

• Today was a happy day to five of us! We went to the U.S. Embassy in Sao Paulo and we got our visas! :D

finO and finP - Still Trying to Confirm our Biobricks

• We ran an agarose gel of yesterday's PCRs product.

• We couldn't obtain even a single amplified fragment! =(

• Why can the transformed cells grew in the media (that means they actually have the AMP resistence), but they haven't our inserts into it's plasmids?

• Our advisors suggested us that, since we digested our plasmid vector with XbaI and SpeI (X sticky ends can come together with S sticky ends), it recircularized without the introduction of our inserts.

• Therefore, we must perform the dephosphorylation of our digested vector. That may prevent it from recircularizing without the insert introduction.

Marcelo

PY Promoter - Colony-PCR screening

• We selected 5 white colonies of each transformation we did yesterday to perform a colony-PCR screening. In this PCR we used the M13 primers of pGEM vector. In those colonies that have the vector with our fragment inserted this pair of primers would amplify a fragment with XX bp (PY1) and XX bp (PY2).

• We analyzed the results of our PCR in an agarose gel:

CeiB: Searching the right colony

• We decided to search in the plate possible cells with the right DNA insert. We made 15 colony of each plate. Unfortunately we didn’t get again.

Ane

YeastGuard

Biofusion vector - Electroelution

• <p style=”text-align:justify;”> Considering that we need more vector to perform the ligation reactions, we electroeluted more vector from the ADH1 biobrick previously digested with XbaI and SpeI (Protocol 12).

Taís