Hi all,
I am currently trying to introduce a mutant gene and sub clone the wild type gene from pcDNA3.0 into pcDNA3.1.
After each gel purification, from PCR product to enzyme digestion, I have run a gel to make sure that it is still a single clear band so that when I do the ligation, I can be sure that it is as clean as possible without any contamination.
The ligation occurs at 16 degree overnight followed by 65 degree 10 mins. The total volume of the ligation is 10ul, with 50ng of vector and either 8.3ng, 55.86ng (mutants) or 65.15ng (wt) of insert.
The overnight culture (16hrs) on the ampicillin agar plate with DH5a (Thermo Fisher) shows that there was only 1 clone in the plate for the wt and nothing for the mutants.
Why does the transformation efficiency is so low? Is it possible that I have destroy the DH5a when handling them?
I have a lot more cloning to do so I really need to figure this out. Thanks!
Your ligation conditions seem alright.
I recommend you do a positive transformation control, with any pure plasmid, on another aliquot of your DH5a cells. This way, you will at least determine if your problem comes from your cells or from the DNA you are using. Using 50-100ng of pure plasmid, you should expect several thousands of transformed clones (nearly confluent growth).
If this test reveals your DH5a cells are fine (highly competent), then you may begin optimizing further your transforming DNA and ligation conditions. For that, I usually work with 100-200 ng total DNA with a 3:1 ratio of insert:vector, in a total volume of 10ul. I ligate at room temperature 30mins or at 16C overnight. I then transform 50ul of competent cells with 5ul of that ligation mixture.
Hope it helps !
Martin's answer is pretty good. There are quite a few similar questions where this has been discueed out in great detail: https://www.researchgate.net/post/Problems_with_BL21DE3_pLysE_bacteria_transformation
As a very-quick fix, for low-efficiency ligation's I generally recommend plating more cells. You can gently spin down the DH5a cells after the recovery step (around 1K RPM for 5min on a benchtop centrifuge), then gently re-suspend and plate the entire pellet. That will get your the maximum number of colonies from your transformation.
Also, I generally follow NEB's protocol, in case your protocol differs substantially: https://www.neb.com/protocols/0001/01/01/high-efficiency-transformation-protocol-c2987
Hi Mathew,
You haven't mentioned how much ligation mix you use for transforming your DH5a cells. From what I read in your description, you have 5.8-11.0 ng DNA in per microliter of your ligation mix.
While transforming DH5a, or any other hosts for that matter, you should not use more than 1 microliter of the ligation mixture. As ligase, and other buffer components may interfere in transformation. More amount of ligation mixture hampers the transformation.
You can also try reducing the antibiotic concentration of the agar plates. For example, if you are using 100ug/ml Ampicillin in your agar plates, try using 50ug/ml Ampicillin instead. This trick works many times...
One more thing, if DH5a is not working, then you may consider Top10 cells for transformation. They give much better results than DH5a when it comes to transforming ligation mixtures.
Last option, which is quite risky and I usually don't recommend, is ethanol precipitation of ligation mixture, followed by 70% EtOH wash twice and dissolving in same amount of nuclease-free water and use in transformation.
Hope this helps...
Good luck :-)
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