Difference between revisions of "E coli colony PCR"
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| − | |H<sub>2</sub>O|| | + | |H<sub>2</sub>O|| 11.54 μl |
|- | |- | ||
| − | | Taq PCR Buffer 10X || | + | | Taq PCR Buffer 10X || 1.5 μl |
|- | |- | ||
| − | | dNTPs (10 mM each)|| 0. | + | | dNTPs (10 mM each)|| 0.6 μl |
|- | |- | ||
| − | | MgCl<sub>2</sub> (50mM)||0. | + | | MgCl<sub>2</sub> (50mM)||0.6 μl |
|- | |- | ||
| − | |Primer Fwd (10 μM) || 0. | + | |Primer Fwd (10 μM) || 0.3 μl |
|- | |- | ||
| − | |Primer Rev (10 μM) || 0. | + | |Primer Rev (10 μM) || 0.3 μl |
|- | |- | ||
| − | | Taq polymerase (5U/μl) || 0. | + | | Taq polymerase (5U/μl) || 0.16 μl |
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| || 52°C || 30 sec | | || 52°C || 30 sec | ||
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| − | | || 72°C || 1 min 30 sec | + | | || 72°C || 1 min 30 sec (or ~1kb/min) |
|- | |- | ||
|1X || 72°C || 7 min | |1X || 72°C || 7 min | ||
Revision as of 16:59, 4 January 2021
This is a protocol to do PCR reactions on E. coli cells, directly after a transformation. (See here for colony PCR for yeast cells)
- Prepare mastermix
- Divide 19μl/sample over PCR reaction tubes/wells
- Prepare cells in water
- Add cell mixture to the mix
Re-suspend cells for each colony in 50μl DNAse free water and then add 1μl of this mix to the reaction.
| Do sequencing reaction: | |
|---|---|
| H2O | 11.54 μl |
| Taq PCR Buffer 10X | 1.5 μl |
| dNTPs (10 mM each) | 0.6 μl |
| MgCl2 (50mM) | 0.6 μl |
| Primer Fwd (10 μM) | 0.3 μl |
| Primer Rev (10 μM) | 0.3 μl |
| Taq polymerase (5U/μl) | 0.16 μl |
Run using program (bart -> colony PCR or cPCR)
| Number of cycles | Temperature | Duration |
|---|---|---|
| 1 X | 94°C | 2 min |
| 35X | 94°C | 30 sec |
| 52°C | 30 sec | |
| 72°C | 1 min 30 sec (or ~1kb/min) | |
| 1X | 72°C | 7 min |
| 7°C | Inf |
