Sunday, July 31
Once again tried tranformation into competent cells. Used 2 protocols 1st protocol from New England Biodabs High Efficiency Protocol for C2987 cells Added 1 ul of each of material from tubes 1, 3, 2B, 2, 1B, (dot or scribble),4A, 5A, 5B to 50 ul of competent cells (was not enough competent cells to do all tube samples) Added control PUC19 to one of the competent cell vials, and put no dna into the second competent cell vial.
Left on ice for 30 minutes. heat shocked tubes 2 at a time for 45 secs. Placed on ice for 5 minutes. be Pipetted 950 ul of room temp SOC into each tube put in warm room shaker, shook for 1 hour
Also Used a Protocol from Tom, from Chung, Niemela, and Miller "One-step preparation of competent Escherichia coli: Transformation and storage of bacterial cells in the same solution," Proc Natl. Acad. Sci Vol 86, pp 2172-2175, April 1989 Added 1ul of each material from tubes 3B and 4A to 900 uL of 2xTSS. No dna and PUC19 were use as controls put into warm room, and shook for 1 hour
Plated all (both protocols) using hockey stick dipped in 80% ethanol, to smear 100 uL of each sample and at 17:30 plates were put into warm room
Excess material was put in freezer, labelled "C2987 Transformation" or "TSS Transformation" with today's date, in green rack
Total hours 5.5 SS