I carry out ligation reaction at 22 deg C for one to two hours using T4 DNA ligase and then directly use the reaction mix for transforming DH5 alpha cells. I would like to know if inactivating T4 DNA ligase before transforming will increase the efficiency of transformation?
The paper Rajeev is referring to was specific to electroporation. I have never notice much if any effect of inactivating ligase with chemically competent cells, although I have noticed a small effect with removal of ligase buffer by doing an ethanol precipitation. However this was only a few fold effect. If you are not getting many colonies I would first focus on preparation of your competent cells and your transformation method.
To my knowledge a ligase has no negative effect on the transformation, therefor an inactivation is not necessary and increase the efficiency.
But, you should keep in mind, that the ligation buffer can have a negative effect on a transformation performed by electroporation, and that you can concentrate the ligation mixture with a purification and thus increase the transformation efficiency.
Yes, it may increase the efficiency.
Below is given a paper where it has been shown that transformation fold is increased when Ligase is inactivated. Transformation was done by electroporation.
Transformation of Escherichia coli increases 260-fold upon inactivation of T4 DNA ligase. Michelsen BK. Anal Biochem. 1995 Feb 10;225(1):172-4.
(http://www.ncbi.nlm.nih.gov/pubmed/7778774)
I usually perform ligation overnight in fridge (4 degrees of Celsius). works good.
The paper Rajeev is referring to was specific to electroporation. I have never notice much if any effect of inactivating ligase with chemically competent cells, although I have noticed a small effect with removal of ligase buffer by doing an ethanol precipitation. However this was only a few fold effect. If you are not getting many colonies I would first focus on preparation of your competent cells and your transformation method.
Ligase will definitely decrease the efficiency of both chemical transformation and electroporation and that is why they recommend you heat inactivate it first. That being said, you can often get colonies without heat inactivating it, and you only need one positive right? I typically use the thermocycler to do my ligations for 1h with a 5min heat inactivation step at the end (but our machine isn't in high demand!). With electroporation, the high salt in the ligation buffer might cause arching if you use too much in your mixture. So if necessary you might need to spin column purify your ligation mixture. Check out Sambrook's Molecular Cloning. It's a good resource.
To my knowledge a ligase has no negative effect on the transformation, but with electroporation, the high salt in the ligation buffer might cause arching if you use too much in your mixture. So if necessary you might do ethanol precipitation before transformation or use chemical transformation.
No. However, if you use the promega 2X rapid ligation buffer and make ligation reaction at 4C overnight, followed by using 5 microliters of ligation reaction to transform DH5 alpha cells. This really increase the transformation efficiency.
I don't know, if T4 DNA ligase has a negative effect on transformation. But i often ligate inserts into a plasmid and transformate competent e.coli with these ligations. If i have really low efficiecies, i perform the ligation over night instead of one or two hours. I get satisfying results with this little aklteration.Perhaps it can also help you to increase your efficiecy. Another possibility for low efficiencies is, that your competent cells do not work proper. Did you buy them or make the on your own? Did you test the efficiency of your competent cells with other plasmids like e.g. amaxa-gfp or puc19?
I am not sure if inactivating the ligase will help, but another tact to try is altering your ligation to overnight at 4oC. We regularly use DH5a cells for transformation, and we have good success with transformation following a 12-14 hour ligation at 4oC with the T4 ligase. We do not inactivate the ligase.
We do not electroporate for transformation. If you do not either, one other thing to consider is the amount of time you heat shock the cells during your transformation, as this can also drastically influence your success. Try two different methods to test the efficiency, I would recommend a low time/higher temp and a longer time/ lower temp. We usually try 37oC for 30secs (or even 45) and 42oC for 20 secs with DH5alphas.
It would be good to heat inactivate DNA Ligase, before transformation. In the process of Ligation, DNA ligase joins two DNA strands (or circulation of linear DNA) by forming phosphodiester bond. If you dont heat inactivate the Ligase after ligation reaction, some of the enzyme will be close to DNA stands (even at 42C - during transformation step) - which could decrease the transformation efficiency a bit (no need to take it too seriously). Usually, while performing transformation, we will not purify the ligation mixture after ligation, so better to heat inactivate the ligase. Heat will cause the structural changes in the Ligase, leading it to dissociate from the DNA. This ligase dissociated DNA will more efficient in the transformation process (a little affect on overall transformation efficiency).
I don't know the answer, but the NEB quick ligase protocol advises against heat inactivation of the ligase before transformation.
https://www.neb.com/protocols/1/01/01/quick-ligation-protocol
Hi, you can try with on column purification of your ligations, although I think that it depends what kind of T4 ligase you use as well- a standard or modified one, etc. Do you use the T4 from the kit? try to decrease/increase the time and according to time the T°C of the ligations. Michael Benedik tip about the competent cells and their Transformation is as well a good starting point for optimisation...
In my opinion I think inactivate T4 DNA ligase is not involve with transformation. The factor that effect transformation efficiency is buffer or salt in buffer. So try to clean your DNA and use suitable buffer before transformation and perfect competent cell is important for transformation.
to increase the efficiency of transformation, there is a very efficient protocol described in "A novel method for increasing the transformation efficiency of Escherichia coli—application for bacterial artificial chromosome library construction.Heng Zhu and Ralph A. Dean Nucl. Acids Res. (1999) 27 (3): 910-911". in this protocol, autors use tRNA DURING PRECIPITATION OF LIGATION PRODUCT WITH ETHANOL. In fact, tRNA is not a simply carrier but seems to change the topology of the dna increasing the efficiency of transformation. Using this method you may increase the efficiency for a small plasmid (pgemt...) by 400 times. I used currently and it's very efficient.
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