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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) | |
| - | + | *'''1.2uL''' MgCl<sub>2</sub> | |
| - | + | *'''0.4uL''' dNTPs | |
| - | + | *'''200nM''' final concentration of each primer VF2 and VR (0.2uL) | |
| - | + | *'''0.2uL''' Taq enzyme | |
| - | + | *''template DNA'' | |
| - | (note: the total volume of PCR is 20µL) | + | (note: add ddH<sub>2</sub>O to 20µL, 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 | ||
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== 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 | + | *'''0.4 uL''' MgCl<sub>2</sub> |
| - | + | *'''2 uL''' PCR Buffer | |
| - | 0.2 uL Taq | + | *'''2 uL''' colony culture |
| - | + | *'''0.2 uL''' Taq enzyme | |
| + | *'''15.8 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 | ||
Latest revision as of 16:11, 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 VF2 and VR (0.2uL)
- 0.2uL Taq enzyme
- template DNA
(note: add ddH2O to 20µL, 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
- 0.4 uL MgCl2
- 2 uL PCR Buffer
- 2 uL colony culture
- 0.2 uL Taq enzyme
- 15.8 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
