Team:UNIPV-Pavia/Notebook/Week1Sep

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*We inoculated a single colony from K116002 streaked plate in 1 ml of LB + Amp. We incubated the inoculum at 37°C, 220 rpm overnight.
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*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).
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Revision as of 18:59, 27 September 2009

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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

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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.

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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).

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September, 3rd

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September, 4th

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September, 6th

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