Thursday, August 25

From 2011.igem.org

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Dr Liz : Lab Opening Time 3:00

Jessica Hobson 2p.m.- close

• Desirae: 2:30pm-close

• Bivor: 12 pm-close

• Travis 7 PM - 10 PM

• Steve 7 PM-close

1. Reviewing colony PCR from 8/4/11

Cells were transformed on 7/31/11 with site-directed mutagenesis products. Colony PCR was performed on 8/4/11 with colonies from 3B and 4A and the gel was run on 8/17/11. The DNA ladders looked good on the gel, but here were no PCR products on the gel, even for the positive control pET-Taq plasmid, suggesting that the PCR reaction didn’t work.

Possible future steps: Repeat PCR for the positive control pET-Taq until the positive control works. (Possibilities: change annealing temperature, change template concentration, use new dNTPs).

Alternatively, rather than doing colony PCR, we can grow up the cells containing the plasmids, purify the plasmids with a Qiagen kit and digest the plasmids with PstI. We picked colonies from the 3B and 4A plates and grew them overnight in 3 ml LB+ampicillin. Plasmids will be purified using the Qiagen kit on Friday and PstI digestions will be performed on Sunday.

2. Reviewing colony PCR from 8/17/11

Cells were transformed on 8/4/11 with site-directed mutagenesis products. Colony PCR was performed on 8/17/11 with colonies from 3B and 4A. The DNA ladders looked good on the gel, but there were no PCR products on the gel, even for positive control pET-Taq plasmid, suggesting that PCR reaction didn’t work.

Future steps: Troubleshoot as above.

3. Hardware update: WE HAVE A PCR MACHNE!!!!!! Made of tin sample container filled with mineral oil. Ramp rate is slow, however, and needs to be improved. Need to look at thermal conductivity of various materials such as salt and powdered aluminum, reduce the volume of liquid in the container. How much does ramp rate matter? Future directions: Try making a sample holder, fully test and measure the temperature ramp rates, test different conductive fluids, controller works but doesn’t cool yet needs a PCB, and Travis is working on writing an interface program in the Python language. Drs. Burkett and Scheifele will work on finding a good template/primer combination that can be used to test the PCR machine.

4. Biobrick parts: Composite parts were transformed in Towson lab-result was no colonies on the plate. We confirmed that the correct plate of parts was used, so there must have been a problem with the transformation at Towson. Transformation was repeated at CCBC, there were colonies on the plate (chloramphenicol resistant). Colonies were picked and plasmids were purified. DNA was confirmed by restriction digest to be correct. So we now have nice DNA that we can cut the promoter/RBS parts out of. Also finished making the plasmid preps of the terminator part, so we can cut the terminator out of that plasmid as well.

Future steps: team members worked with Dr. Burkett to determine how to cut and paste the promoter, RBS, coding sequence and terminator together in one vector using the PlasmaDNA program.