96-WELL PCR

Mike Deyholos (08 Mar 00)

1. Check whether the buffer contains MgCl_2.

2. Combine the appropriate reagents in a sterile tube:

If the buffer DOES NOT have MgCl₂

1 plate 2 plates

4125 ml 8250 ml autoclaved nanopure water

500 ml 1000 ml 10X buffer without MgCl₂

300 ml 600 ml MgCl₂ (25mM)

48 ml 96 ml dNTP mix (25mM each)

30 ml 60 ml Sigma Taq pol (5U/ml)

1.5 ml 3 ml forward primer (1mM)

1.5 ml 3 ml reverse primer (1mM)

If the buffer HAS MgCl₂

1 plate 2 plates

4425 ml 8850 ml autoclaved nanopure water

500 ml 1000 ml 10X buffer with MgCl₂

48 ml 96 ml dNTP mix (25mM each)

30 ml 60 ml Sigma Taq pol (5U/ml)

1.5 ml 3 ml forward primer (1mM)

1.5 ml 3 ml reverse primer (1mM)

3. Aliquot the above reagents and template into a 96-well PCR plate (e.g. Greiner 651501); seal with a wax sheet and place in a pre-heated thermocycler programmed as follows:

95’C 4 min.

95’C 30 sec. }

57’C 30 sec. } 42 cycles

72’C 50 sec. }

72’C 7 min.