Week from June 29th, to June 30th, 2009

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June, 29th

• We read that using primers VR/VF2 to PCR B0010 will result in excess bands, as documented by Samantha Burke in http://partsregistry.org/Problems_with_PCR_using_VR/VF2 . Maybe our extra bands were due to unwanted annealing between VR primer and B0015 which contains B0010.

• In the future we will perform the screening of the ligations either through digestion or colony PCR taking carefully into account of their expected length and their potential non-specific primer binding sites.

• We picked 4 colonies from A8 plate and 10 colonies from A9 plate (both stored at +4°C) and infected 1 ml of LB + Amp. We incubated the cultures at 37°C, 220 rpm for 5 and 1/2 hours.

• Glycerol stocks for the grown cultures.

• We re-filled the remaining 250 ul of bacterial culture with 5 ml of LB + Amp and incubated the cultures overnight at 37°C, 220 rpm. Tomorrow we will repeat the screening on these plates through miniprep/digestion!

June, 30th

• Miniprep for the 13 overnight cultures.

• Digestion E-P for 800 ng of the purified plasmids.

• Electrophoresis for the 14 digestions.

Digestion results for A8 and A9:

• Gel results:

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