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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]]''' | ||
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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''' | + | *'''1.2uL''' Mgcl<sub>2</sub> |
*'''0.4uL''' dNTPs | *'''0.4uL''' dNTPs | ||
*'''200nM''' final concentration of each primer | *'''200nM''' final concentration of each primer | ||
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*'''5 uL''' colony culture | *'''5 uL''' colony culture | ||
*'''0.2 uL''' Taq | *'''0.2 uL''' Taq | ||
| - | *'''10.4 uL''' | + | *'''10.4 uL''' H<sub>2</sub>0 |
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 14:09, 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
