How to Ligate DNA

We will follow the suggested protocol provided by Promega's 2X Rapid Ligation protocol. In order to do this, you will need to calculate the amount of insert you need with this formula:

X ng of insert = (2) (bp insert) (50 ng linearized plasmid-) ÷ (size of plasmid in bp)

The 2 in the numerator takes into account the fact that you want to have a 2:1 ratio of insert to vector. You want to use 50 ng of plasmid for a typical ligation.

Thaw the frozen 2X buffer at room temperature. After the first thawing, it is best if you aliquot the 2X buffer in smaller volumes to avoid repeated freeze/thaw cycles. If a precipitate is present, vortex the solid DTT until it is back in solution. This usually takes 1 - 2 minutes.

Set up the following 10 µl ligation reaction (if your DNA is too dilute, you can scale up to 20 µL ligation volume and transform all 20 µL. But try to minimize the ligation volume.

Digested Vector (50 ng)	1 µl
Insert (2:1 molar ratio insert:vector)	(< 3) x µl
Sterile water	3µl- x µl
2X ligation buffer	5 µl
3 units T4 DNA ligase (keep cold)	1 µl
Total Volume	10.0 µl

Incubate for 5 minutes at room temperature. From this point, you may either freeze the ligation or go directly to transformation.

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