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Summary Quickchange Site-directed mutagenesis Protocol
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A detailed explanation of how site-directed mutagenesis works, the different steps involved, and why we carry out those steps. Created by a graduate Biochemistry student who is now studying Biological Sciences at Cambridge University. This is designed to be used for all levels of learning, from GCS...

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  • July 28, 2024
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  • 2023/2024
  • Summary

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Site-directed Mutagenesis
QuickChange Site-Directed Mutagenesis


Introduces specific changes to DNA sequences


PRIMER DESIGN
1 Design a complementary pair of primers containing the desired mutation, each 25-
45 nucleotides long, with the mutation in the middle. Primers are short sequences
of nucleotides that initiate DNA synthesis. By placing the mutation in the centre of
the primers, you ensure that both strands of the DNA can be amplified, allowing for
efficient incorporation of the desired mutation during PCR.



PCR AMPLIFICATION
2 The PCR reaction should contain:
Template DNA: 10-100 ng
Forward Primer: 125 ng
Reverse Primer: 125 ng
dNTPs (10 mM each): 1 µL
PCR buffer (10X): 5 µL
DNA Polymerase: 1 µL (2.5 units)
Water: Up to 50 µL total volume



PCR CYCLING
3 Denaturing separates the DNA strands whilst annealing allows the primers to
bind to their complementary sequences on the template. DNA polymerase then
synthesises new strands by adding nucleotides (dNTPs) to the primers.


STEP TEMPERATURE TIME CYCLE NO.


Initial denature 95ºC 30 seconds 1


Denaturation 95ºC 30 seconds


Annealing 55-65ºC 1 minute 18-25


Extension 68-72ºC 1 minute per kb


Final Extension 72ºC 10 minutes 1





DPN1 DIGESTON

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