During the ligation step we are going to mix the linerized vector and the insert together
and add a T4 DNA ligase. The ligase will fuse their ends forming a circular plasmid.
Ligation protocol:
1. Ligation mix o 9 µl ddH2O
o 2 µl 10X ligation buffer o 2 µl of plasmid digested with restriction
enzymes A and B o 6 µl of insert digested with restriction
enzymes A and B. For the ligation control, use 6 µl of water instead of insert
o 1 µl DNA ligase
2. Split ligation mix into two tubes 10 µl each
3. Incubate tube 1 at room temperature for 15 min
4. Incubate tube 2 at 16 oC for 24 hours
Let's set up the ligation reaction. Here on the bench I have:
* Double distilled water * A tube with cut vector
* A tube with cut insert * An aliquot of ligase buffer - remember that
the ligase buffer has to be aliquoted to avoid freeze-thawing
* An empty tube for vector plus insert * An empty tube for control
* And the T4 DNA Ligase
I am going to set up two reactions, one with vector and insert and one with vector alone
for control.
In the Vector plus Insert tube I am going to combine:
* 9 µl of water * 2 ul of cut vector
* 6 ul of cut insert * 2 µl of ligase buffer
*
And 1 µl of DNA ligase
And as always, I mix it up and down with a pipette.
The control tube is pretty much identical, the only difference is that it contains water
instead of insert.
In the control tube I am going to put: * 15 µl of water, it's 9 + 6 instead of insert
* 2 ul of cut vector * 2 µl of ligase buffer (we are skipping
the insert) * 1 µl of DNA ligase
And the mixing step.
Now I am going to split the contents of ligation tubes into two aliquots. Here I have two new
tubes, one of them I will label as Vector Plus Insert, another one as Vector Control.
And I will transfer 10 ul from each of the ligation tubes into its replica.
I am going to leave these two tube on the bench for 15 minute room temperature ligation,
and I will put the other two tubes in a 16 oC incubator overnight.
If tomorrow I will not get any colonies from the 15 minute ligation, I will repeat the
transformation step with the tubes I kept at 16 oC.