Team:Calgary/15 July 2010


Thursday July 15, 2010

Jeremy's restriction enzyme on pRFP EcoRI/HindIII and XbaI/HindIII to remove pRFP from its original plasmid


  • Today I did a Colony PCR of the the I0050 + B0034 Construct with the primers that Emily provided me with.

PCR conditions: Stage 1: 94 degrees Celcius Stage 2; Step 1: 94 degrees 1 minute Stage 2; Step 2: 55 degrees 45 seconds Stage 2; Step 3: 72 degrees 1 minute 15 seconds Stage 3: 72 degrees Holding Temperature: 4 degrees Celcius

  • I have also assisted Alex with his Gel electrophoresis of the CpxP colony pcr. We are hoping to see a band around

the length of 150 bp.


  • Today I reconstructed K239000 with I13507 and I13504, then K135000 with I13507 and I13504 with proper plating. I also did a restriction enzyme digest of pRFP with EcoRI and HindIII and also XbaI and HindIII. And a gel was run of the restriction enzyme products. Also 4 tubes were prepared for I0500+B0034+E0040 to test inducing arabinose.


Ran all three gels of the PCR reactions. The reaction containing the highest magnesium ion concentration yielded PCR in all 12 reactions. The DNA found was of the right size (~200)bp.


  • Today I did more research about high and low copy plasmid. Dave came up with a new idea of constructing the transcription/translation circuit and Emily and I spoke about it to finalize a strategy.


Today, I did more calling of companies to apply for sponsorships as well as writing the application for Transalta sponsorship. We also did PCR purifications of the CpxP promoters but ended up with very low concentrations and very high levels of contamination with proteins and RNA. Because of this, we decided to redo the PCR purifications but ended up with little difference in concentration and contaminations. Regardless, we did restriction digests and left these overnight to place into plasmids tomorrow.


  • Today I diluted the F and R BBK primers and the malE-N-F and R primers. On Monday we'll do a test PCR to make sure that both of these sets are working. I also finalized primer sequences for biobricking malE and malE31 with signal sequence. On Monday I will be looking into primer seuqneces for the biobricking of malE and malE31 with the signal sequence deletion. Today I also helped Chris make some more AMP plates. We also had a discussion about an improved cloning strategy for the trascription/ translation circuit. Chris, Patrick and I also did a PCR purification of the cpxP promoter PCR product and we set up an overright restriction digest with XbaI and SpeI. We also digested the psb1AK3 and the psb1AC3 vectors with these enzymes in order to get the cpxP promoter into these two vectors. We will antarctic phosphotase, ligate and then transform this part tomorrow.


I ran a gel of the gradient PCRs. There we no bands on the 0.5μL and 1.5μL Mg2+ gradients, but every band on the 2.5μL gradient showed up very well over all temperatures. After analyzing a few samples of the gradient, it's determined that most of them produced about 310-320 ng/μL of product. Along with Chris and Emily, we purified the PCR product and digested them to be ligated into plasmid tomorrow.