Difference between revisions of "PCR"
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(Created page with "For a 25 ul reaction add: :H<sub>2</sub>O 10.5 µl :Taq 2X Master Mix 12.5 µl :Forward primer 0.5 ul 10 µM solution :Reverse primer 0.5 ul 10 µM solution :DNA template 1...") |
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Revision as of 15:30, 16 July 2018
For a 25 ul reaction add:
- H2O 10.5 µl
- Taq 2X Master Mix 12.5 µl
- Forward primer 0.5 ul 10 µM solution
- Reverse primer 0.5 ul 10 µM solution
- DNA template 1 µl
General PCR cycling conditions are:
- An initial 95 C 30 s
- Then 30 cycles of:
- 95 C 20 s melting
- annealing temperature (this varies with the primers used, usually between 55 C and 65 C although some primers can be lower) 40s
- 70 C 1 m extension (this can be raised to 5 m for several kbp long segments)
- With a final extension of 70 C for 5 minutes.
