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USTC/Notebook/PCR&Colony PCR
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2. add the following components: | 2. add the following components: | ||
| - | + | - 2µL PCR buffer (rock gently after thawing, quick spin before use) | |
| - | + | - 1.2uL Mgcl2 | |
| - | + | - 0.4uL dNTPs | |
| - | + | - 200nM final concentration of each primer | |
| - | + | - 0.2uL Taq enzyme | |
| - | + | - template DNA | |
(note: the total volume of PCR is 20µL) | (note: the total volume of PCR is 20µL) | ||
| Line 26: | Line 26: | ||
== colony PCR == | == colony PCR == | ||
| - | 0.2 uL primer #1 (to 25uM) | + | - 0.2 uL primer #1 (to 25uM) |
| - | 0.2 uL primer #2 | + | - 0.2 uL primer #2 |
| - | 0.4 uL dNTPs | + | - 0.4 uL dNTPs |
| - | 2 uL Buffer | + | - 2 uL Buffer |
| - | 5 uL colony culture | + | - 5 uL colony culture |
| - | 0.2 uL Taq | + | - 0.2 uL Taq |
| - | 10.4 uL H20 | + | - 10.4 uL H20 |
perform PCR with an initial heating step at 94C for 5 minutes followed by 25-35 cycles of 30sec at 94C, 30sec at 55C and 1Kb/min at 72C,and 72C for 10min | perform PCR with an initial heating step at 94C for 5 minutes followed by 25-35 cycles of 30sec at 94C, 30sec at 55C and 1Kb/min at 72C,and 72C for 10min | ||
Revision as of 13:44, 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: - 2µL PCR buffer (rock gently after thawing, quick spin before use) - 1.2uL Mgcl2 - 0.4uL dNTPs - 200nM final concentration of each primer - 0.2uL Taq enzyme - template DNA (note: the total volume of PCR is 20µL)
3. make sure reaction tubes are properly capped before placing in thermocycler
4. perform PCR with an initial heating step at 94C for 5 minutes followed by 25-35 cycles of 30sec at 94C, 30sec at 55C and 1Kb/min at 72C
colony PCR
- 0.2 uL primer #1 (to 25uM) - 0.2 uL primer #2 - 0.4 uL dNTPs - 2 uL Buffer - 5 uL colony culture - 0.2 uL Taq - 10.4 uL H20
perform PCR with an initial heating step at 94C for 5 minutes followed by 25-35 cycles of 30sec at 94C, 30sec at 55C and 1Kb/min at 72C,and 72C for 10min
