Note: After two failures, we repeated this using new enzyme and increased primer amounts. Original recipe was:
* 32ul water
* 5ul 10x buffer
* 1ul 50mM MgSO4
* 0.5ul 1uM Fwd_Primer
* 0.5ul 1uM Rev_Primer
* 10ul Template (5 ng)
* 1ul Pfx enzyme
You will use ~5ng of intact plasmid template in 5ul
50ul total reaction volume
- 17.1ul water
- 1.5ul 10mM dNTPs
- 5ul 10x buffer
- 1ul 50mM MgSO4
- 10ul 1uM Fwd_Primer
- 10ul 1uM Rev_Primer
- 5ul Template (5 ng)
- 0.4ul Pfx platinum enzyme (ThermoFisher #11708013)
A second set of samples was prepared using the 10x PCRx Enhancer solution included with the enzyme:
- 14.6ul water
- 1.5ul 10mM dNTPs
- 5ul 10x buffer
- 1ul 50mM MgSO4
- 10ul 1uM Fwd_Primer
- 10ul 1uM Rev_Primer
- 5ul Template (5 ng)
- 0.4ul Pfx platinum enzyme (ThermoFisher #11708013)
- 2.5ul 10x PCRx Enhancer
— hotstart enzyme, reactions can be assembled at RT.
Primer combos TP53_MISO_Fwd1/TP53_MISO_Rev1 - 309 bp - 30 sec elongation
TP53_MISO_Fwd2/TP53_MISO_Rev2 - 567 bp - 45 sec elongation
TP53_MISO_Fwd3/TP53_MISO_Rev3 - 493 bp - 30 sec elongation
Thermocycler program
95*C - 2 min. (initial denature)
61*C - 30sec (annealing)
68*C - 30sec - 1min (extension - 1min/kb)
95*C - 15 sec
61*C - 30sec
68*C - 30sec - 1min
95*C - 15 sec
59*C - 30sec
68*C - 30sec - 1min
2 cycles
95*C - 15 sec
57*C - 30sec
68*C - 30sec - 1min
2 cycles
95*C - 15 sec
55*C - 30sec
68*C - 30sec - 1min
2 cycles
95*C - 15 sec
53*C - 30sec
68*C - 30sec - 1min
2 cycles
95*C - 15 sec
51*C - 30sec
68*C - 30sec - 1min
2 cycles
95*C - 15 sec
49*C - 30sec
68*C - 30sec - 1min
2 cycles
95*C - 15 sec
47*C - 30sec
68*C - 30sec - 1min
2 cycles
95*C - 15 sec
45*C - 30sec
68*C - 30sec - 1min
13 cycles
95*C - 15 sec
45*C - 30sec
68*C - 5min
4*C - HOLD
30 total cycles
