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)

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• 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, 11st

• We transformed the overnight ligations of B5 and B6. 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.

August, 12nd

• 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!

August, 13rd

August, 14th

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