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

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August, 10th

• Digestion for:

• 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:

• 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.

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:

• 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).

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).

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).

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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