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Site-Directed Mutagenesis on a Minicircle using Overlap Extension PCR

PCR Success Story #22

Project Description

Generate double mutants in a minicircle vector with contains very few unique restriction sites.

(paid service)

The goal was to combine mutations in a minicircle vector coding for the genes of a Human Papilloma Virus Strain.

The researcher supplied us with 3 single mutant vectors, in which we needed to introduce a downstream mutation that would abolish the start codon for a second gene encoded within the first gene but in a different reading-frame.

Project Details

Client: McGill University
Date: April 1rst, 2017
Type of experiment: Create double mutants in a minicricle vector
DNA Polymerase:  FastPfu FLY

Competitor: N/A

Vector Restriction Digest

Full Restriction Digest of the Backbone Vector

1rst Digestion (60 min)

ddH2O : to 50 ul
10x buffer : 5 ul
Vector : 2 ug
AgeI-HF : 1.5 ul
BsiWI : 1.5 ul

DNA Clean-Up

Follow the instructions of FavorPrep GEL/PCR Purification Kit or FavorPrep MicroElute GEL/PCR Purification Kit and elute the DNA in 40 ul EB buffer.

2nd Digestion (15 min)

ddH2O : 3 ul
10x buffer : 5 ul
Eluate : 40 ul
AgeI-HF : 1 ul
BsiWI : 1 ul

DNA Clean-Up
  1. Mix 10 ul of 6x DNA loading buffer with your sample and load on a 0.7 % agarose gel prestained with GelStain.
  2. Cut out the band using a clean razor blade and follow the instructions for FavorPrep GEL/PCR Purification Kit. Elute the DNA in 40 ul EB buffer.

PCR used to Generate a Mutation and Create Overlapping DNA Fragments

Fast & Steep PCR to generate LEFT and RIGHT fragments

Left 1, 2 or 3 (1.3 kb) fragments, each amplified from their respective parent template containing only either one of the three mutations, and thee Right 123 (0.6 kb) DNA fragment, used to add a second and common mutation to any of the 1rst mutations, were amplified by Fast & Steep PCR.

PCR Setup :

ddH2O : to 35 ul
5x buffer: 7 ul
dNTPs (2,5 mM) : 2.8 ul
F primer (100 uM) : 0.14 ul (14 pmol)
R primer (100 uM) : 0.14 ul (14 pmol)
*Minicircle : 300 ng
FastPfu FLY : 0.7 ul (1.75 u)

PCR Cycling: 7 cycles (Ta = 62 °C)