Oct 05, 2021

Public workspaceOverlap & Gibson ligation

DOI
dx.doi.org/10.17504/protocols.io.byrspv6e
  • 12021 iDEC NEFU_China
  • NEFU_China 2021
Shuning Guo
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DOI: dx.doi.org/10.17504/protocols.io.byrspv6e
Protocol Citation: Shuning Guo 2021. Overlap & Gibson ligation. protocols.io https://dx.doi.org/10.17504/protocols.io.byrspv6e
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: October 04, 2021
Last Modified: October 05, 2021
Protocol Integer ID: 53778
Keywords: Overlap, Gibson assembly
Abstract
This protocol is used to lagate two pieces of DNA together without digesting the fragment with restriction endonucleases.
Materials
DNA fragments
Primers
ClonExpress II One Step Cloning Kit (Vazyme)
2×High Fidelity Master Mix (MCLAB)
Nanodrop
Thermocycler
Water bath
DdH2O

Safety warnings
Please wear gloves for the experiment, don't try to touch the lid after PCR program initiation.
Before start
Set up a small box with ice, put DNA and enzymes on it.
Prepare the water bath to 37℃ to have Gibson assembly.
Ligation of two DNA fragments by using cases below.
Step case

Overlap PCR
10 steps

Preparation of linearized vectors
Preparation of linearized vectors
Select an appropriate cloning site on the vector that will be linearized.
Vector linearization: the linearized vector can be obtained by digesting the circular vector with restriction enzymes or by reverse PCR.
PCR of the inserts DNA fragments
PCR of the inserts DNA fragments
Amplify the insert DNA fragments with homologous sequences (for homologous recombination) of vector-upstream or -downstream by PCR using high fidelity DNA polymerase.
Calculate amount and ratio of linearized vectors and Inserts
Calculate amount and ratio of linearized vectors and Inserts
Detect DNA concentration of linearized vectors and inserts by Nanodrop.
Calculation of the amount of vectors:
Molar ratio of vector to insertion is 1:1
Recombination & PCR
Recombination & PCR
Set up the following reaction on ice (50μl):

AB
Forward Primer (10 μM)1μl
Reverse Primer (10 μM)1μl
Fragment1(vector)X
Fragment2(insertion)Y
2×High Fidelity Master Mix (MCLAB)25μl
ddH2OAdd to 50μl
The primer is used to amplify recombinant DNA fragment/circular DNA.

Program the thermocycler as follows:
AB
TemperatureTime
95/98℃5 min
95/98℃30 s
Tm-3~5℃30 s
72℃1kb/min
72℃5~10 min
16℃∞
Repeat 30 times in 3-5 steps

Use the palm centrifuge to mix the solution in PCR tube.
Put the PCR tube into the thermocycler and Run the program.
Using agarose gel electrophoresis to confirm if correct construct was present.