Simply Cloning - Chapter 6 - Ligation

Simply Cloning is a video manual for making DNA constructs.
Chapter 6 describes how to ligate DNA fragments together using a T4 DNA ligase.

Narration Script:

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.