PCR screening

This protocol enables you to determine after ligation whether a particular colony contains empty vector or insert.

  • Dip a pipette tip in a colony and then resuspend the cells in 100ul of water. Vortex to ensure resuspension.

  • Make up the following master mix (sufficient for 19 reactions)

169ul H2O

20ul buffer

10ul W-1 (from the Invitrogen Taq+W-1)

6ul MgCl2

2ul each primer stock (100uM)

2ul dNTPs (stock of 10mM of each dNTP)

2ul Taq

The primers for this PCR should be specific to the vector (eg for T7 and T7term). This means that you will amplify across the MCS - a small insert will imply empty vector, a large insert (of the correct size) will imply ligation of the target.

  • Take 9ul of master mix and add 1ul of resuspended colony for the following PCR

Initial denaturation

94oC for 2min

35 cycles of

denaturation: 94oC for 30s

annealing: 50oC for 30s

extension: 72oC for 1min/kbp

Final extension

72oC for 7 min

  • Add loading dye to the PCR tube and run on a gel. Look to see the length of the amplified fragment.

  • For those fragments of the correct size, add 10ul of the colony/water mix, add to 5ml of LB media and grow overnight in a shaking incubator at 37oC for a miniprep.

All this should fit in one day, so from looking at your ligated plates in the morning you should be able to grow that evening overnight cultures only of those colonies which you think contain insert.