Gama:Site directed mutagenesis
Overview
Site directed mutagenesis protocol by PCR
Materials - plasmids and primers
Template DNA
Plasmid with sequence to be mutated
Plasmid without that sequence (control)
Primers
A sense primer has to contain the desired mutation included in the bases that are complementary to the existing sequence.
An anti-sense primer has to contain the complementary bases of the existing sequence. Example:
Existing sequence
5′ggacgcaagctgtaatgctctagacgtta-------------tgtggtcattgtgtcaccgcaggacattga 3′
3′cctgcgttcgacattacgagatctgcaat-------------acaccagtaacacagtggcgtcctgtaact 5’
Desired sequence
5′ggaTCcaagctgtaatgctctagacgtta-------------tgtggtcattgtgtcaccgcaggacattga 3′
3′cctAGgttcgacattacgagatctgcaat-------------acaccagtaacacagtggcgtcctgtaact 5′
5′ggaTCcaagctgtaatgctcta→
3′cctgcgttcgacattacgagatctgcaat-------------acaccagtaacacagtggcgtcctgtaact 5′
5′ggacgcaagctgtaatgctctagacgtta-------------tgtggtcattgtgtcaccgcaggacattga 3′
<-cagtggcgtcctgtaact 5′
-Primers must be diluted from the stock to a final concentration of 10 µM.
Procedure
Mix preparation
Prepare a mix for each of the mutations by adding the following components to an Eppendorf tube:
- 84 µL of distilled water
- 15µL of KOD Hotstart Buffer 10X
- 9 µL of MgSO4 25mM
- 15 µL of dNTPs 2mM
- 4,5 µL of primer Fw 10 µM
- 4,5 µL of primer Ver 10 µM
Notes
Unfreeze mix components slowly on ice.
Make sure the two primers do not contact before you are ready to add the mix to the template DNA by pipetting each to opposite sides of the Eppendorf tube wall.
Then, add 3 µL of KOD Hotstart enzyme and the mix is ready to use.
Shortly before addition to DNA tubes, mix gently.
DNA preparation
Dilute your DNA stock solutions to final concentration of 10 ng/ µL and then add 5 µL of each to PCR tubes.
Pippete 45 µL of desired mix to each two DNA containing tubes (target and control). Total volume of each reaction will now be 50 µL.
DNA concentration in PCR reaction may be changed, according to your needs. In such cases, change the volume of DNA described above and correct the water volume added to the corresponding mix.
PCR protocol
Place the PCR tubes appropriately in the device and run the following PCR protocol:
- Initialization step - 95°C - 2’
- Denaturation step - 95°C - 20’’
- Annealing step - 55°C - 10’’
- Elongation step - 70°C - 3’
- Final elongation - 70°C - 3’
Perform at least 24 cycles between denaturation and elongation steps. Annealing temperature can be adapted depending on primers melting temperature (at least 5°C below the lowest primer melting temperature).
After PCR reaction, take 5 µL and run an agarose gel to check if template DNA was properly amplified.
Template DNA digestion
Digest the remaining 45 µL of the PCR result by adding 1 µL of DpnI enzyme and incubate at 37°C for 1 hour (this enzyme only digests methylated DNA).
Mutated DNA amplification
Transform competent bacteria with PCR amplified plasmids and purify the DNA. Mutations confirmation can be achieved by sequencing the resultant DNA .
