*<p style=”text-align:justify;”>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).</p>
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*<p style=”text-align:justify;”>We analyzed the results of our PCR in an agarose gel:</p>
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:
GEL
YeastGuard
Biofusion vector - Electroelution
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).
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