TITLE: Overlap & Gibson ligation
AUTHORS: Shuning   Guo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is used to lagate two pieces of DNA together without digesting the fragment with restriction endonucleases.</div></div>

[STEPS]
?. Ligation of  two DNA fragments by using cases below.
?. [Preparation of linearized vectors]
Select an appropriate cloning site on the vector that will be linearized.
?. [Preparation of linearized vectors]
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]
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]
Detect DNA concentration of linearized vectors and inserts by Nanodrop.
?. [Calculate amount and ratio of linearized vectors and Inserts]
Calculation of the amount of vectors:The optimal amount of vector for the recombination with ClonExpress II is 0.03 pmol, while the optimal amount of insert is 0.06 pmol (molar ratio of vector to insertion is 1:2), as roughly calculated as follows: The optimal mass of vector = [0.02 × number of base pairs] ng (0.03 pmol) The optimal mass of insert = [0.04 × number of base pairs] ng (0.06 pmol)
?. [Recombination]
Dilute linearized vectors and inserts before recombination to make sure the loading accuracy. The volume of each component loaded should be no less than 1 μl.
?. [Recombination]
Set up the following reaction on ice:Linearized VectorsX μlInsertsY μl5 × CE II Buffer4 μlExnase II2 μlddH2OAdd to 20 μl
Linearized VectorsX μlInsertsY μl5 × CE II Buffer4 μlExnase II2 μlddH2OAdd to 20 μl
?. [Recombination]
Incubate at 37℃ for 30 min and immediately place the tube at 4℃ or on ice.
?. [Preparation of linearized vectors]
Select an appropriate cloning site on the vector that will be linearized.
?. [Preparation of linearized vectors]
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]
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]
Detect DNA concentration of linearized vectors and inserts by Nanodrop.
?. [Calculate amount and ratio of linearized vectors and Inserts]
Calculation of the amount of vectors:Molar ratio of vector to insertion is 1:1
?. [Recombination & PCR]
Set up the following reaction on ice (50μl):Forward Primer (10 μM)1μlReverse Primer (10 μM)1μlFragment1(vector)XFragment2(insertion)Y2×High Fidelity Master Mix (MCLAB)25μlddH2OAdd to 50μl
Forward Primer (10 μM)1μlReverse Primer (10 μM)1μlFragment1(vector)XFragment2(insertion)Y2×High Fidelity Master Mix (MCLAB)25μlddH2OAdd to 50μl
?. [Recombination & PCR]
Program the thermocycler as follows:TemperatureTime95/98℃5 min95/98℃30 sTm-3~5℃30 s72℃1kb/min72℃5~10 min16℃∞
TemperatureTime95/98℃5 min95/98℃30 sTm-3~5℃30 s72℃1kb/min72℃5~10 min16℃∞
?. [Recombination & PCR]
Use the palm centrifuge to mix the solution in PCR tube.
?. [Recombination & PCR]
Put the PCR tube into the thermocycler and Run the program.
?. [Recombination & PCR]
Using agarose gel electrophoresis to confirm if correct construct was present.
?. [Recombination]
Use the palm centrifuge to mix the solution in PCR tube.