Week from June 29th, to July 5th, 2009

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June, 29th

• This week is dedicated to debugging: we wanted a pure plasmid for A8 and A9 ligations, but the screened colonies seemed to have the correct plasmid + unwanted extra bands (see previous week).

• 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: all the lanes showed the correct plasmid, but also the unwanted one. We kept A8-3 and A9-7 for further study.

• 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 - it cannot be easily seen in the image above, but unfortunately it was there!).

• 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

July, 1st

• Sequencing results for BOL1 BioBrick: sequence was correct!

• Miniprep for:
  • A8 (for dilution/transformation)
  • A9 (for dilution/transformation)

• We transformed 10 pg of A8 and A9 miniprepped DNA in TOP10, after a proper quantification with Nanodrop and dilution. We plated transformed bacteria and incubated them overnight at 37°C. (we will call these transformations "10 pg plates")

• We prepared 0.5 l of LB + Amp.

• We infected 5 ml of LB + Amp with 10 ul of T9002 glycerol stock. We incubated the culture overnight at 37°C, 220 rpm.

• We also picked a random colony from A10 plate and infected 1 ml of LB + Amp. We incubated this inoculum at 37°C, 220 rpm for 5 and 1/2 hours. Then we prepared a glycerol stock and re-filled the remaining 250 ul of culture with 5 ml of LB + Amp to grow an overnight culture (for sequencing).

Preparation of experiment with Tecan F200

• We diluted 1:1000 the cultures of:
  • J23100
  • J23101
  • J23118
  • A1
  • A2
  • A7
  • E0240

• We incubated the diluted cultures at 37°C, 220 rpm for 3 hours.

Experiment with Tecan F200

• Download Protocol

July, 2nd

• Miniprep for A10 and T9002. We sent purified DNA to BMR Genomics for sequencing.

• A8 and A9 (10 pg plates) plates showed colonies!(a few, as expected) We picked 3 colonies for A8 plate and 3 colonies for A9 plate to infect 5 ml of LB + Amp. We incubated the six inocula at 37°C, 220 rpm overnight. Tomorrow they will be miniprepped and screened with digestion to check if the extra band had been lost!

Preparation of experiment with Tecan F200

• aTc stock preparation.

• We diluted 1:1000 the cultures of:
  • A8
  • A9 (X2)
  • E0240

• We incubated the diluted cultures at 37°C, 220 rpm for 3 hours. After 2 hours, one of the two A9 culture was induced with 75 ng/ml aTc and incubated again under the same conditions as before.

Experiment with Tecan F200

• Download Protocol

July, 3rd

• Miniprep for the overnight cultures (taken from the two 10 pg plates).

• Digestion E-P for the purified plasmids (under the same conditions as the experiment of June 30th).

Digestion results for A8 and A9 (10 pg plates): A8-3 and A9-1 colonies were kept.

• Gel results: the plasmids seemed to be pure (except from A8-1 in which the unwanted insert is easily visible). We decided to keep:
  • A8-3
  • A9-1
• glycerol stocks and to validate them with sequencing. We will call them "A8pg" and "A9pg".

• Tecan F200 data analysis.

• Team meeting.

Items of a serious team meeting: plastic glasses, an empty tray of sweets, a paper boat (??) and prof. Magni saying something very serious:)

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