3 More DNA ligation Tips
A while back, I wrote an article on 5 DNA ligation tips that could improve the efficiency of your cloning procedures. It proved to be quite a popular article so here are another 3 tips that might make your ligations even better!
1. Change ligase brand. All T4 DNA ligase preps are not equal. Many researchers report that they obtain different results with different ligases, but there does not seem to be a consensus on which is the best. So why not find out for yourself? If you are having ligation problems, call up your local sales reps and ask for a free sample of their ligase. Then try out the ligases side-by-side to see which works best for you.
2. Be careful with PEG. One of the tips I gave in my previous article was to add polyethylene glycol (PEG) to your ligation mixtures. The rationale behind using PEG is that is this hydrophobic molecule “crowds” the aqueous solution of the ligation mix, effectively increasing the concentration of the DNA and ligase, making their reaction more efficient. For this reason, PEG is a component of the buffers of the quick ligation kits that are on the market.
However, the downside to PEG is that it can reduce transformation efficiency. To prevent this, ensure that you:
- Do not heat inactivate ligations containing PEG. Many users heat inactivate the ligase by incubation at 65degC for 15 minutes after the ligation, but in reactions containing PEG this reduces the transformation efficiency.
- Do not use extended ligation times in PEG-supplemented reactions. Most quick ligation kits recommend incubation times of the order of 5-60 minutes. Going above this will reduce the transformation efficiency.
- Do not use a high concentration of PEG. DNA ligation efficiency is improved by PEG concentrations of 5-15% but transformation efficiencies are reduced when PEG is greater than 5%. So, use 5% PEG in the transformations.
3. Precipitate using a tRNA as a carrier. This week I came across this 1999 Nucleic Acids Research paper that shows plasmid ligation efficiencies can be increased by 400-fold, simply by ethanol precipitating the ligation mixture in the presence of yeast tRNA. The effect seems to be robust and the authors showed that it is specific to tRNA - the same effect was not observed when ds or ss herring sperm DNA was used as the carrier. They speculate that the tRNA is altering or stabilising the DNA in a topological form that is more “transformable”. Whether this is true is anyone’s guess, but this seems like a good thing to try and we are testing it in our lab at the moment.
If you try this, be sure to let us know your results, or if you have any other ligation tips share them here….
molecular biology
Others advise not to use tRNA as a general tool for precipitation:
http://www.fermentas.com/catalog/reagents/glycogen.htm
Hengen PN.
Carriers for precipitating nucleic acids.
Trends Biochem Sci. 1996 Jun;21(6):224-5.
PMID: 8744357
Thanks Kurt.
It seems that the two articles you cite there state that tRNA has two disadvanatges when used as a carrier.
The fermentas article states that it does not assist precipitation as well as glycogen does, and the TIBS article mentions that tRNA carry-over can affect certain downstream applications like hybridizations.
In the protocol I mentioned, the main role of the tRNA is though to be to stabilise the DNA in a more transformable form so in this case any inefficiency in assisting with ligation is not really too much a of a concern. And, since it doesn’t seem to affect transformation efficiencies, it doesn’t seem like tRNA carry-over will be a problem either.
But, the proof is in the pudding - I am in the process of checking whether precipitating the ligation with tRNA actually does increase overall efficiency… so watch this space.
Thanks for flagging this up though. I was not aware of this TIBS archive - there are some great articles in there. Anyone interested in reading them should go to this URL: http://www-lmmb.ncifcrf.gov/~pnh/readme.html
In the related methods, an article that seems to be a new technique for DNA transformation in bacteria.
http://nar.oxfordjournals.org/cgi/content/abstract/gkm710v1
I think I will also try the tRNA tip soon in my lab…
Cheers.
We have always ethanol precipitated our ligations before transformation with glycogen and ammonium acetate. Then a wash with 70% Ethanol. This works very well to clean them up for electroporation also.