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Team:University of Ottawa/22 July 2008
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Today on the Lab
Matt
- Gel of Digestion of PTP2 Ligation Product
- I attempted several digestion's with EcoRV/EagI, EcoR, EagI, plasmid. I had many unexpected bands with each sample some of which were above 10 kb. Im thinking its contamination.
- I then attempted another digestion with PstI, EcoRV, AatII. Again I didn't get any of the expected band sizes.
Chris
- Ligation Efficiency Experiment
- To help determine the issues with our ligations, ran two separate ligations using two separate methods.
- For sample A, digested DQ232598B with EagI, followed by PCR cleanup
- For sample B, digested DQ232598B with EagI, followed by denaturation of enzymes
- For control, used water in place of DNA sample and performed ligation as usual, with no procedure afterwards.
- Removed samples of A and B after each step.
- Ran completed ligation against samples collected from each step as well as controls on a 1% gel for 40 minutes.
- Sample B ligated correctly, while A did not. It was subsequently determined that NEBuffer is more successful for ligation than ligase buffer. The age of our ligase buffer came into question and all old stocks of it were disposed of.
- Inoculation of AtCRE
- Inoculated transformed competent cells in liquid LB for miniprep the following day. Ran one control tube without a colony.
