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USTC/Notebook/PCR&Colony PCR
From 2008.igem.org
(Difference between revisions)
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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) | + | - 2µL PCR buffer (rock gently after thawing, quick spin before use) |
| - | - 1.2uL Mgcl2 | + | - 1.2uL Mgcl2 |
| - | - 0.4uL dNTPs | + | - 0.4uL dNTPs |
| - | - 200nM final concentration of each primer | + | - 200nM final concentration of each primer |
| - | - 0.2uL Taq enzyme | + | - 0.2uL Taq enzyme |
| - | - template DNA | + | - template DNA |
(note: the total volume of PCR is 20µL) | (note: the total volume of PCR is 20µL) | ||
| - | |||
3. make sure reaction tubes are properly capped before placing in thermocycler | 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 | 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 | ||
| Line 30: | Line 28: | ||
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 | |
2 uL Buffer | 2 uL Buffer | ||
5 uL colony culture | 5 uL colony culture | ||
Revision as of 13:43, 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
