Week from August 31st, to September 6th, 2009

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August, 31st

• We inoculated:
  • 10 colonies from B5new2 plate in 4 ml of LB + Kan for screening;
  • B8-5 (8 ul from glycerol stock) in 4 ml of LB + Kan (for purification process);

• We sent A15-2 purified DNA (stored at -20°C) to BMR Genomics for sequencing.

Experiment with Tecan F200

• Description
• Purpose:
• Materials & Methods
• Protocol
• Results

September, 1st

• We received sequencing results for:
  • A16-4: sequence ok!
  • A14-3: sequence had a deletion in C0012, as we expected because of A11-2 sequencing results, previously received;
  • B8-5: sequence wrong in VF2 and good in VR;
  • A17: chromatogram was good, but ligation failed (R0011 was not in the insert).

• COMMENTS after sequencing results:
  • now we have a new aTc sensor (A16) to test together with A9;
  • B8 has to be repeated or purified, we will try both the approaches;
  • A14 has to be repeated using A11-3, which has a consistent sequencing result;
  • A17 is going to be ligated today (this time using gel extraction).

• Glycerol stocks for the 10 B5new2 grown cultures.

• Miniprep for B5new2 (10 samples) and for B8-5.

• Digestion for:
  • B5new2 (10 samples) for screening (E-P cut);
  • B8-5 for purification from gel (E-P cut);
  • R0011 (stored at -20°C) for A17new ligation (S-P cut)
  • E0240 (stored at -20°C) for A17new ligation (X-P cut)

• Medium-size gel for B5new2(E-P); small-size gel for B8-5(E-P), R0011(S-P) and E0240(X-P).

• Medium gel results: B5new2 clones all showed the correct length for ligated plasmid! we decided to keep B5new2-3 to continue the assemblies and B5new2-9 as a backup copy. Both plasmids were sent to BMR Genomics for sequencing.

• Small gel results: R0011(S-P) and E0240(X-P) were cut and purified through gel-extraction, while B8-5 could not be cut because the bands were not correct: maybe the heaviest band noticed on August 24th was a non-completely digested fragment, not a correct-size insert. We decided to throw B8-5 glycerol stock away.

• Ligation: A17new = R0011(S-P) + E0240(X-P) in pSB1A2

• We incubated ligation reaction at 16°C overnight.

• We inoculated:
  • B5new2-3
  • B6-3
  • A4
  • F2620MIT1
  • A11-3

• We streaked a single colony LB + Amp agar plate using K116002 glycerol stock. We incubated the plate at 37°C overnight.

September, 2nd

• Miniprep for:
  • B5new2-3
  • B6-3
  • A11-3
  • A4
  • F2620MIT1
  • E0240 pellet (stored at -20°C)

• Digestions:
  • B5new2-3(E-X)
  • B6-3(E-X)
  • A11-3(S-P)
  • A4(E-S)
  • F2620MIT1(E-S)
  • E0240(X-P)

• Gel run, cut and band purification for all the samples.

• Ligations:
  • B7new = A4(E-S) + B5new2-3(E-X) in pSB1AK3
  • B8new = A4(E-S) + B6-3(E-X) in pSB1AK3
  • B9 = F2620MIT1(E-S) + B5new2-3(E-X) in pSB1AK3
  • B10 = F2620MIT1(E-S) + B6-3(E-X) in pSB1AK3
  • A14L = A11-3(S-P) + E0240(X-P) in pSB1A2

• We incubated the five reactions at 16°C overnight.

• Transformation/plating for A17new ligation.

• We inoculated a single colony from K116002 streaked plate in 1 ml of LB + Amp. We incubated the inoculum at 37°C, 220 rpm for about 6 hours. Then we prepared a glycerol stock for it and re-filled the remaining part of the culture with 5 ml of LB + Amp. We incubated this culture overnight (37°C, 220 rpm).

September, 3rd

• Miniprep for K116002 overnight culture. We used 1 ul of this sample to transform TOP10 in order to have the pH sensor in the same strain as the reference promoter (i.e. A2). We also sent purified DNA to BMR Genomics for sequencing.

• A17new overnight plate showed only one colony...easy screening for this plate! Colony PCR/electrophoresis for this sample. We stored this colony in 100ul of LB + Amp and incubated it at 37°C waiting for the end of the reaction.

• Gel results: the blank was not so clean, but the only colony was positive:) We inoculated the colony in 5 ml of LB + Amp to grow an overnight culture (37°C, 220 rpm).

• We transformed these ligations:
  • B7new 1:40 on Kan
  • B8new 1:40 on Kan
  • B9 1:40 on Kan
  • B10 1:40 on Kan
  • A14L 1:20 on Amp

September, 4th

September, 6th

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