Team:UNIPV-Pavia/Notebook/Week2Aug

From 2009.igem.org

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Week from August 10th, to August 16th, 2009

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August, 10th

  • Digestion for:
B1-13(E-S)(X2) B2-5(E-S)(X2) B3-5(E-X)(X2)
B4-2(E-X)(X2)
  • Gel run for all of them (only 1 ul for B1-13(E-S)(X2) in order to check the length)

Gel run for B1, B2, B3 and B4.

  • We saw a couple of spurious bands in B3...but we ignored them and cut the single band with expected length.
  • Band cut/purification for:
B2-5(E-S)(X2) B3-5(E-X)(X2) B4-2(E-X)(X2)
  • Ethanol precipitation with sodium acetate for B1-13(E-S)(X2).
  • Ligation:
    • B5 = B1(E-S) + B4(E-X) in pSB1AK3
    • B6 = B2(E-S) + B3(E-X) in pSB1AK3
  • We incubated the ligation reactions at 16°C overnight.

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August, 11th

  • We transformed the overnight ligations of B5 and B6 (20 pg). We plated the transformed bacteria on LB agar plates + Kan and incubated the plates at 37°C overnight.


  • Digestion for:
BOL1(E-S) R0010(E-X) F2620MIT1(E-S)
K112808(E-X)
  • Gel run/cut/purification for all of them. All the bands were present at the right size!
  • Ligation (20 ul final volume) for:
    • A11 = BOL1(E-S) + R0011(E-X) in pSB1A2 (we call it A11 again, because the previous A11 had a deletion ad we threw it away)
    • A15 = F2620MIT1(E-S) + K112808(E-X) in pSB1A2
  • We incubated the ligations at 16°C overnight.


  • M9 supplemented medium preparation (with glycerol). This first attempt showed precipitations...we will try tomorrow to dissolve MgSO4 and CaCl2 in ddH2O very slowly.



Preparation of experiment with Tecan F200

  • We inoculated 10 ul of A2 glycerol stock and a single colony of B0033 from its native plate in 5 ml of LB + Amp.
  • We incubated the cultures overnight (37°C, 220 rpm).

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August, 12th

  • We transformed 1 ul of A11 and A15 ligations in TOP10. We plated on LB + Amp agar plates and incubated them in the morning.
  • After about 9 hours the plates showed many colonies! we picked three single colonies from each plate and infected 5 ml of LB + Amp. We incubated these inocula at 37°C, 220 rpm overnight to grow up cultures to screen.


  • We picked 7 colonies from B5 and B6 overnight plates and infected 1 ml of LB + Kan. We incubated these 14 inocula at 37°C, 220 rpm for 6 hours and then we prepared glycerol stocks.
  • We re-filled the remaining 250 ul with 5 ml of LB + Kan to grow overnight cultures to screen.


  • We repeated M9 supplemented medium preparation and we finally had it without precipitations!



Preparation of experiment with Tecan F200

  • We diluted 1:100 the overnight cultures of A2 and B0033.
  • We incubated the diluted cultures for 5 hours (37°C, 220 rpm).


Preparation of experiment with Tecan F200 (for the following day!)

  • We inoculated 10 ul of A2 glycerol stock in 5 ml of LB + Amp.
  • We incubated the culture overnight (37°C, 220 rpm).

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August, 13th

  • Glycerol stocks for:
    • A11 (3 samples)
    • A15 (3 samples)
  • Miniprep for:
    • B5 (7 samples)
    • B6 (7 samples)
    • A11 (3 samples)
    • A15 (3 samples)
  • Digestion screening for all the samples:
    • B5(E-P)
    • B6(E-P)
    • A11(S-P)
    • A15(E-P)
  • Medium-size gel for B5 and B6; small-size gel for A11 and A15.

Screening digestion for B5 and B6 (both cut E-P).

Screening digestion for A11 (S-P) and A15 (E-P).

  • Gel results:
    • B5 - the first colony was surely wrong, while the others showed the right size for ligated plasmid, but also two smaller unexpected bands...
    • B6 - all the colonies showed the right size for ligated plasmid, but also two smaller unexpected bands...
    • A11 - the first colony was a false positive, while A11-2 and A11-3 showed the expected length for ligated plasmid.
    • A15 - all the 3 samples had the expected length for a correct insert and vector.
  • We will repeat the screening for B5 and B6 changing the enzymes to understand something more.


  • Overnight digestion for the 14 miniprepped samples of B5 and B6 (HindIII-EcoRI).


Preparation of experiment with Tecan F200 (for tomorrow!)

  • We inoculated 10 ul of A11-2, A11-3, A15-1, A15-2 and A15-3 in 5 ml of LB + Amp.
  • We incubated the inocula overnight (37°C, 220 rpm).



Preparation of experiment with Tecan F200

  • We diluted 1:100 the overnight culture of A2.
  • We incubated the diluted culture for 5 hours (37°C, 220 rpm).


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August, 14th

  • Miniprep for:
    • A11-2
    • A11-3 - low yield, to do again.
    • A15-1
    • A15-2 - low yield, to do again.
    • A15-3


  • Electrophoresis for the overnight digestion (B5 and B6 cut HindIII-EcoRI).

Debugging digestion for B5 and B6 (HindIII-EcoRI).

  • Gel results: all the samples showed the expected bands for ligated plasmids, but also MANY other unwanted bands...



Preparation of experiment with Tecan F200

  • We diluted 1:100 the overnight cultures of A11-2, A11-3, A15-1, A15-2 and A15-3 in 5 ml of LB + Amp.
  • We incubated these dilutions for 5 hours (37°C, 220 rpm).



Experiment with Tecan F200

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