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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)

DNA Assembly using PCR Overlap Extension

Fast & Steep Overlap Extension PCR for 3 mutations

Fast & Steep OE-PCR 1.9 kb

Left 1, 2 or 3 (1.3 kb; 10 ul each) and Right 123 (0.6 kb; 10 ul) DNA fragments were fused into a 1.9kb DNA molecule under 30 min using Fast & Steep PCR.

PCR Setup :

ddH2O : to 40 ul

5x buffer: 8 ul

dNTPs (2,5 mM) : 3.2 ul

F primer (10 uM) : 1.6 ul (16 pmol)

R primer (10 uM) : 2 ul (16 pmol)

LEFT DNA : 10 ul

RIGHT DNA : 10 ul

FastPfu FLY : 0.8 ul (2 u)

PCR Cycling: 8 cycles (Ta = 57 °C)

Total run time: 26 min

Gel load : 2 ul / lane

Cloning of the Overlapped DNA into the Linearized Vector

Seamless Assembly of a DNA Fragment and a Linearized Vector

ddH2O : to 5 ul
2x assembly mix : 2.5 ul
Linearized vector : 0.4 ul
Insert : 1.1 ul

Incubation : 20 min at 50 °C

Transformation of the Fully Assembled Minicircles into Competent Cells

Plasmid Transformation into Trans2-Blue Chemically Competent Cells

  1. Thaw 50 ul of cells on ice.
  2. Add 1.5 ul of the pEASY-Uni reaction mix
  3. Gently flick the tube twice, then put on ice for 30 min
  4. Heat Shock for 30 s at 42 °C in a water bath.
  5. Put on ice for 2 min.
  6. Add 450 ul of room temperature SOC or LB media.
  7. Shake at 200 rpm at 37 °C for 45 min.
  8. Spread 200 ul on a pre-warmed kanamycin-selective LB-agar plate.
  9. Incubate O/N at 37 °C

* We typically spread 60-100 ul on LB-agar plates, but this particuliar minicircle vector has very poor transformation efficiency in all strains tested.

Clone Validation by Restriction Analysis and Sanger Sequencing

Plasmid Minipreps and Restriction Analysis

Plasmid Minipreps

Two to four colonies per plate were picked and inoculated in 2.5 ml LB media and selective for kanamycin-resistant bacteria.

An aliquot of 850 ul was reserved for the glycerol stock and the remaining bacteria (≤ 1.65 ml) served for plasmid DNA extraction using FAPDE-300 from Favorgen.

The DNA was eluted in 50 ul EB solution.

Restriction Analysis

ddH2O : to 10 ul
10x CS buffer : 1 ul
Plasmid DNA : 2 ul
Bsu36I : 0.3 ul
NdeI : 0.3 ul

Incubated 15 min at 37 °C.

One or two clones were sent at the Plateforme du Séquençage du CHUL for Sanger sequencing. Sequences were aligned and chromatographs analyzed using Snapgene.

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