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Team:TU Darmstadt/Protocols/mutagenic PCR
From 2012.igem.org
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=== Working principle === | === Working principle === | ||
| - | [[File:Mutagene_pcr_plasmid_v2.png|200px|thumb|right|Figure 1.]] | + | [[File:Mutagene_pcr_plasmid_v2.png|200px|thumb|right|'''Figure 1.'''A shematic representation of mutagenic PCR]] |
The basic procedure requires the synthesis of a short DNA primer. This synthetic primer contains the desired mutation and is complementary to the template DNA around the mutation site so it can hybridize with the DNA in the gene of interest. The mutation may be a single base change (a point mutation), multiple base changes, deletion or insertion. The single-stranded primer is then extended using a DNA polymerase, which copies the rest of the gene. The copied gene contains the mutated site, and is then introduced into a host cell as a vector and cloned. Finally, mutants were selected by restriction digestion with a disered restriction enzyme. | The basic procedure requires the synthesis of a short DNA primer. This synthetic primer contains the desired mutation and is complementary to the template DNA around the mutation site so it can hybridize with the DNA in the gene of interest. The mutation may be a single base change (a point mutation), multiple base changes, deletion or insertion. The single-stranded primer is then extended using a DNA polymerase, which copies the rest of the gene. The copied gene contains the mutated site, and is then introduced into a host cell as a vector and cloned. Finally, mutants were selected by restriction digestion with a disered restriction enzyme. | ||
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| - | ===Themocycler | + | ===Themocycler program=== |
# T= 98°C 1 min | # T= 98°C 1 min | ||
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# T= 72°C 8 min | # T= 72°C 8 min | ||
# Hold 4°C | # Hold 4°C | ||
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Latest revision as of 22:36, 26 September 2012
Contents |
Mutagenic PCR
This site-directed mutagenesis is used to change particular base pairs in a piece of DNA.
Working principle
The basic procedure requires the synthesis of a short DNA primer. This synthetic primer contains the desired mutation and is complementary to the template DNA around the mutation site so it can hybridize with the DNA in the gene of interest. The mutation may be a single base change (a point mutation), multiple base changes, deletion or insertion. The single-stranded primer is then extended using a DNA polymerase, which copies the rest of the gene. The copied gene contains the mutated site, and is then introduced into a host cell as a vector and cloned. Finally, mutants were selected by restriction digestion with a disered restriction enzyme.
Primers
primers designed for this PCR:
Mixtures
| Reaction mix | Volume/Mass |
|---|---|
| 5x Phusion HF buffer | 5µL |
| dNTP´s | 1µL |
| DMSO | 1,5 µL |
| DNA | 5,25,50 ng |
| Primer fwd | 125 ng |
| Primer rev | 125 ng |
| Phusion-Polymerase | 0,5 µL |
| H2O | ad 50 µL |
Themocycler program
- T= 98°C 1 min
- T= 63°C 0,5 min
- T= 72°C 3 min
- go to 2 rep 15
- T= 72°C 8 min
- Hold 4°C
