*<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>
+
*<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. We analyzed the results of our PCR in an agarose gel:</p>
-
+
-
*<p style=”text-align:justify;”>We analyzed the results of our PCR in an agarose gel:</p>
+
[[Image:py_gel_3.png|400px|center]]
[[Image:py_gel_3.png|400px|center]]
+
+
*<p style=”text-align:justify;”>The expected size for PY1 + pGEM amplified with M13 primers is 390 bp.</p>
+
+
*<p style=”text-align:justify;”>The expected size for PY2 + pGEM amplified with M13 primers is 332.</p>
+
+
*<p style=”text-align:justify;”>Unfortunately, just the colonies with PY1 displayed bands with the expected size in the gel. Since the sequence of PY1 is more complete than the sequence of PY2, we decided to not try again with PY2 and use just the colonies that have pGEM vector with PY1.</p>
+
+
*<p style=”text-align:justify;”>So we selected 2 colonies with PY1 + pGEM and inoculated them in liquid LB-AMP at 37
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. We analyzed the results of our PCR in an agarose gel:
The expected size for PY1 + pGEM amplified with M13 primers is 390 bp.
The expected size for PY2 + pGEM amplified with M13 primers is 332.
Unfortunately, just the colonies with PY1 displayed bands with the expected size in the gel. Since the sequence of PY1 is more complete than the sequence of PY2, we decided to not try again with PY2 and use just the colonies that have pGEM vector with PY1.
So we selected 2 colonies with PY1 + pGEM and inoculated them in liquid LB-AMP at 37
CeiB: Searching the right colony
<p style=”text-align:justify;”>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).
"
