DNA ligations can be frustrating. Sometimes they just don’t work, for no obvious reason. My top 5 DNA ligation tips will help to improve the efficiency of your ligation reactions and hopefully give a better cloning success rate.
1. Aliquot the Ligase Buffer
The ATP in the ligase buffer is essential for the DNA ligation reaction, but is broken down by repeated freeze-thaw cycles. To prevent loss of this essential reaction component between ligations, make aliquots of ligase buffer from the fresh stock provided whenever you buy a new stock of DNA ligase. Make the aliquots small enough so that you can throw them away after use, and before you make the aliquots, ensure that the buffer is completely de-frosted and mixed well.
2. Heat the DNA just Prior to Ligation
When setting up a sticky (cohesive) end ligation, pipette the vector and insert fragments first and heat to 65°C for 5 minutes before adding the remaining reaction components. The heating step disrupts any vector/vector or insert/insert sticky end interactions that can interfere with the required vector/insert interaction and reduce the efficiency of the ligation.
3. Adjust the pH
The optimum pH range for the DNA ligation reaction is between 7.6 and 8.0. Depending on how the DNA fragments were prepared, the ligation mixture may have a pH out of this range. It is well worth checking the pH of the ligation mixtures by pipetting approx 0.2 micro-litres of the mix onto narrow range pH paper (e.g. pH 6-8). If required, adjust the pH using 0.2 micro-litre drops of 2M Tris base or HCl.
4. Use Polyethylene Glycol (PEG)
As with any chemical reaction, the speed of the ligation reaction is dependent on the concentration of the substrates. PEG is a hydrophobic molecule that takes up space in the reaction, effectively increasing the concentration of the aqueous reaction components e.g. DNA, ATP and ligase. PEG (e.g. PEG 8000) should be added to a final concentration of around 5-15% .
5. Add a Restriction Enzyme Just Before Transformation
This neat trick can be used to combat high background resulting from uncut vector. If the vector fragment removed during the preparative digest contains a unique restriction site, adding the restriction enzyme to the ligation will selectively digest any uncut vector, preventing it form being transformed. Adding 1 microlitre of the enzyme around 5-10 minutes before transforming should be sufficient.
Originally published on August 24, 2007. Revised and updated on June 1, 2016.
This is the first in a three part series on the transformation of E.coli. By the end of this you should be an expert on E.coli transformation and on which strains to choose for different applications. If you’re already an expert, I hope it’ll be an enjoyable refresher for you. In either case, please comment […]
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