Team:Calgary/21 July 2010


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Wednesday July 21, 2010


Objective: PCR out native MalE/ MalE 31 protein out of E.coli Purpose: Testing Mal E/ Mal E31 primers to get 1188 bp long gene of interest. Primers that are used are gene specific and anneals right on the start of the Mal E gene.

Tube Labels containing 1.0 μL of 10 mM DNA

  • 1- p1H - C1
  • 2- p1H - C1
  • 3- p1H - C2
  • 4- p1H - C2
  • 5- p31H - C1
  • 6- p31H - C2
  • 7- p1ΔSS - C1
  • 8- Master Mix

PCR Conditions

  • Cycle 1: 94°C 2:00 min
  • Cycle 2 * 36 cycles:
    • Stage 1:94°C for 1:00 min
    • Stage 2:52°C for 0:45 min
    • Stage 3:72°C for 1:15 min
  • Cycle 3: 72°C for 10:00 min
    • Holding Temp: 4°C


Today I miniprepped the I0500-B0034 construct from yesterday. One of the constructs gave good concentration and good values for protein and RNA contamination. I also restriction disgested the constructs using EcoRI and PstI. These restriction digests were also ran on 1% agarose gel for 45 minutes at 85 volts. The bands on the gel match our expectation. The insert that was cut using the restriction enzymes was about 1200 bp which means that the I0500 got inserted into the plasmid.


Today, I did Plasmid preparations of the CpxP promoter in both ampicillin-kanamycin and ampicillin-chloramphenicol plasmids. The colony PCR from both of the CpxP promoters was finished overnight and a gel will be run tomorrow on it. As well, I continued to contact different companies like IDT DNA and Abbott International, Generex, AVAC, and Astra Zeneca. We had a meeting in the afternoon where we presented our progress to Dr. Schryvers and Dr. Logan. They gave troubleshooting suggestions for the various wetlab projects we have going on.


Today I ran a PCR of the malE genes that we received from the Betton labs with the malE gene specific primers. Unfortunately we didn't see any amplification, so Raida is going to try that again with some imput from Henry. Unfortunately somebody also miniprepped my overnight cultures for arabinose induction, so that will have to go on hold for a couple of days. Today we also spent a bit of time cleaning up our lab/ classroom spaces. In the afternoon we had a trouble shooting meeting with the whole team. We got some good directions on some new people to contact in terms of our fundraising project as well as some ideas of things to do with the red colonies from our K239000 and K135000 contructs. Today I also made restreaks of our K239000 and K135000 constructs, choosing white/red colonies to compare the two. I also made overnight cultures of I0500-B0034-E0040 in order to try the Arabinose induction again. I also made overnight cultures of the plasmid switched B0034 as well as the psB1AC3 and psB1AK3 vectors (with the ccdb part) from the regitsry and the colonies of cpxP. We left these for overnight growth.


Jeremy and I managed to settle down on a few aspects of the wiki's main page and general layout of other pages. Hopefully, they will be uploaded soon. I also continued to look into high- and low-copy plasmids.

No notebook page exists for this date. Sorry!