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USTC/Notebook/PCR&Colony PCR
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'''[[Team:USTC/Notebook|< Back to Notebook]]''' | '''[[Team:USTC/Notebook|< Back to Notebook]]''' | ||
| + | == PCR == | ||
| + | |||
| + | Briefly, a typical reaction is set up as follows: | ||
| + | |||
| + | |||
| + | 1. set up pre-labeled reaction tubes on ice | ||
| + | |||
| + | |||
| + | 2. add the following components: | ||
| + | - 45µL Platinum PCR SuperMix (rock gently after thawing, quick spin before use) | ||
| + | - 200nM final concentration of each primer (insert calculation short cut here) | ||
| + | - template DNA | ||
| + | (note: the total volume of primers and template can be 0.5 to 20µL) | ||
| + | |||
| + | |||
| + | 3. make sure reaction tubes are properly capped before placing in thermocycler | ||
| + | |||
| + | |||
| + | 4. perform PCR with an initial heating step at 94C for 2 minutes followed by 25-35 cycles of 30sec at 94C, 30sec at 55C and 1Kb/min at 72C | ||
| - | + | == colony PCR == | |
2 uL primer #1 (to 10uM) 2 uL primer #2 0.4 uL DNTPs 2 uL Buffer 5 uL colony culture 0.2 uL Taq 10.4 uL H20 | 2 uL primer #1 (to 10uM) 2 uL primer #2 0.4 uL DNTPs 2 uL Buffer 5 uL colony culture 0.2 uL Taq 10.4 uL H20 | ||
92C 1:30 (92 0:30 50 :30 72 1:30) repeat x30 72 10:00 | 92C 1:30 (92 0:30 50 :30 72 1:30) repeat x30 72 10:00 | ||
Revision as of 13:33, 27 October 2008
PCR
Briefly, a typical reaction is set up as follows:
1. set up pre-labeled reaction tubes on ice
2. add the following components:
- 45µL Platinum PCR SuperMix (rock gently after thawing, quick spin before use)
- 200nM final concentration of each primer (insert calculation short cut here)
- template DNA
(note: the total volume of primers and template can be 0.5 to 20µL)
3. make sure reaction tubes are properly capped before placing in thermocycler
4. perform PCR with an initial heating step at 94C for 2 minutes followed by 25-35 cycles of 30sec at 94C, 30sec at 55C and 1Kb/min at 72C
colony PCR
2 uL primer #1 (to 10uM) 2 uL primer #2 0.4 uL DNTPs 2 uL Buffer 5 uL colony culture 0.2 uL Taq 10.4 uL H20
92C 1:30 (92 0:30 50 :30 72 1:30) repeat x30 72 10:00
