Rao:Ligation

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Overview

Another essential tool in molecular biology. If Taq polymerase and DNA restriction endonucleases are the bricks of cloning, then T4 DNA ligase is the mortar. After digestion of DNA it is essential to put the fragments together when cloning. This is the role of DNA ligases. T4 works by mediating a dehydration reaction between the 3'-hydroxyl and 5'-phosphate of cleaved DNA fragments giving a phosphodiester bond which links the two DNA fragments together.

There are several kits on the market now, in this lab we have bifurcated. Some lab members use NEB T4 DNA ligase (traditional) and some use the Fermentas Rapid Ligation kit.

Materials

  • Appropriate ligase buffers (use the ones that came with your ligase)
  • T4 DNA ligase
  • 0.8 or 1.8 mL centrifuge tubes

Method

NEB T4 ligase

  • x1 uL DNA A
  • x2 uL DNA B
  • 2 uL 10X ligase buffer
  • 18-x1-x2 uL H2O
  • 1 uL T4 ligase

21 uL total volume

Allow to react on your bench for 1+ hr. Some react at the optimal temperature of 16C. Also, some labs leave the reaction in a 4C refrigerator overnight. I have tried all methods, and the last two give no better results in my hands.


Fermentas Rapid Kit

  • x1 uL DNA A
  • x2 uL DNA B
  • 4 uL 5X Buffer
  • 16-x1-x2 uL H2O
  • 1 uL T4 ligase

21 uL total volume

Allow to react on your bench for about 10-20 minutes.

Extras

  • One benefit of the rapid kits are that you can typically directly electroporate reaction mixture (no cleanup required) which can speed things up. However, the rapid kit buffers contain PEG (polyethylene glycol) which is supposed to decrease transformation efficiency.
  • When cloning an insert into a vector, it is typically desirable to have a 3-6:1 insert:vector ratio.
  • If you are having problems with a vector religatiing, it is often useful to dephosphorylate the vector before ligation.

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