Team:TU Darmstadt/Protocols/mutagenic PCR

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Contents 1 Mutagenic PCR 1.1 Working principle 1.2 Primers 1.3 Mixtures 1.4 Themocycler program

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:

• tctA503_Pst1
• tctA505_Pst1
• tctB162_Pst1
• tphC_Pst1

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

1. T= 98°C 1 min
2. T= 63°C 0,5 min
3. T= 72°C 3 min
4. go to 2 rep 15
5. T= 72°C 8 min
6. Hold 4°C