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.

• The 9th picked colony of A9 didn't show any growth.

• Glycerol stocks for the 13 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 13 digestions.

Digestion results for A8 and A9:

• Gel results: all the screened colonies of A8 and A9 showed the ligated insert, but also a small amount of the unwanted insert (non correctly ligated plasmid).

• We decided to keep:
  • A8-3
  • A9-7
• glycerol stocks. 10 ul of them were used to infect 5 ml of LB + Amp and the inocula were incubated at 37°C, 220 rpm overnight. These cultures will be miniprepped and the purified plasmids will be diluted and transformed in TOP10, in order to try to have pure colonies.

Preparation of experiment with Tecan F200

• We prepared 5 ml overnight cultures for:
  • A1 (GFP generator under J23100) - from glycerol stock
  • A2 (GFP generator under J23101) - from glycerol stock
  • A7 (GFP generator under J23118) - from glycerol stock
  • J23100 (containing its own RFP) - from glycerol stock
  • J23101 (containing its own RFP) - from glycerol stock
  • J23118 (containing its own RFP) - from glycerol stock
  • E0240 - from the native plate stored at +4°C

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