Polymerase Chain Reaction Protocol Using Vent DNA polymerase by NEB: FGB 10/99

Date:________ Kit used:________ Oligo’s used:_________

Samples: ____________________________________________

PCR Reaction:

ul per reaction: ul Master Mix: PCR Cycles: Time,Temp:

10X Buffer __5__ _____ One cycle at: Denaturation:

Annealing:

100. mM MgCl2 __0-2 _____ Extension:

10. mM dNTP’s __2__ _____ ____ cycles at: Denaturation:

Annealing:

10uM 3’ oligo __2__ _____ Extension:

10uM 5’ oligo __2__ _____

Vent polymerase __0.5 _____

RT Reaction __2__ _____

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Sterile H2O __36.5 _____

___________________________________

F.V.= 50. use _____ ul of master mix per sample.

Final concentrations: 1x buffer; 2-6mM MgSO4; 400uM dNTP’s; 0.4uM oligo’s; 1U/50 ul Vent.

1. A final volume of 50 ul per sample is suggested. This requires 5 ul of 10X buffer, 0.0ul MgCl2 (2mM is in buffer) or 2 ul of 100mM MgSO4 (8 ul of 25mM), 2.0 ul of 10 mM dNTP's (8 ul of 2.5 mM dNTP’s), 2 ul each of the 10uM stock oligo's (or 1 ul of 20uM stock), 0.5 ul of Vent polymerase, 1.0-1.5 ul of your RT reaction and sterile water for a final volume of 50 ul.

2. On ice, prepare a master mix for one more sample than you are planning to perform.

3. Include all the reagents that are not variables (i.e. RT mix or MgCl2 usually).

4. Bring up to 50 ul then mix well and spin down.

5. On ice, overlay with sterile mineral oil, spin down, set thermocycler and run PCR.

6. Run 15 ul of your PCR products in a 1-1.5% gel with EtBr in 0.5X TBE at 100v.

NOTE:

7. Add 1ul of Taq DNA polymerase and incubate at 72'C for 10 minutes for cloning with TA kit.