Alm:PCR
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Standard PCR reactions
- ~100 ng template
- ~100 pmole each primer
- buffer to 1X
- polymerase
- denature 94-95°C
- anneal 5°C less than lowest primer hyb temp
- extend 1’/kb to be amplified
PCR Master Mix (2.5X)
- 62.5 U/ml Taq DNA Polymerase
- 125 mM KCl
- 75 mM Tris-HCl, pH 8.3
- 3.75 mM Mg(OAc)2
- 500 uM each dNTP
5'-CATTAG can be useful to add to prevent exonuclease activity from degrading recognition sites
