PCR Amplification of Adaptor Ligated cDNA Library (E6114)
Protocol
- Mix the following components in a sterile microfuge tube:
DNA (3–25 ng): variable
Primer 1 (50 μM stock): 10 μl
Primer 2 (50 μM stock): 10 μl
LongAmp Taq: 2X
Master Mix: 250 μl
Nuclease-Free Water: variable
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total volume: 500 μl - Aliquot 125 μl into four PCR tubes.
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Cycle step Temp Time Cycles Nick translation 72°C 20 min 1 Initial denaturation 95°C 5 min 1 Denaturation
Annealing
Extension95°C
62°C
70°C15 sec
15 sec
1 min2–10 Final extension 70°C 5 min 1 Hold 4°C ∞ 1 - Purify DNA sample on one column.