Team:Copenhagen/Protocols
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Protocol for Polymerase Chain Reaction Site-Directed Mutagenesis
We used PCR SDM to remove a Xbal restriction site from the luxCDABE cassette to make it compatible with the Biobrick system.
Primer design
Two complementary primers of 25-30 basepairs are designed.
The chosen sequence should be identical to the template strand, except for the single basepair which is to be mutated.
The melting temperature of the two primers are noted.
PCR reaction
Each sample reaction is prepared in a PCR-tube as indicated below:
- X µL of primer 1 for a total of 125 ng of DNA
- X µL of primer 2 for a total of 125 ng of DNA
- 1 µL of template dsDNA
- 10 µL of Phusion buffer (CONC???)
- 1 µL of dNTPs (CONC???)
- 0.5 µL of Phusion Hotstart enzyme (CONC???)
- X µL of ddH2O to a total volume of 50 microliters
Notes
- The template dsDNA should be complete plasmids obtained an purified from an overnight culture. The length of the plasmid should be known.
- A series of template dsDNA dilutions can be included in the range 5-50 ng of dsDNA per reaction. The remaining concentrations should be held constant.
- A control reaction containing ddH2O instead of template should be included.
| Temperature/°C |
Time/s |
Cycles |
| 98° |
30 |
12 |
| 98° |
10 |
12 |
| Average primer Tm +3 |
20 |
12 |
| 72° |
15 s/kb of plasmid length |
12 |
| 72° |
420 |
|
| 10° |
Remaining time |
|
Treatment towards transformation:
Dpn1 treatment:
- 0.5 µL of Dpn1 (20U/µL) is added directly to each PCR-tube, and the sample is gently mixed by pippeting up and down.
- Each sample is incubated at 37 °C for 60 minutes.
Transformation
See transformation protocol.
Optional:
Restriction site analysis.
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