Greetings!
I am having a problem ligating a 1.5k bp DNA with pMAL and pET28a(+) plasmids. I am certain that my Fast Digest procedure was correctly done because on my gel, I ran double digested, single digested and undigested plasmids and I could see that double digestion was ok.
However, I have tried 2:1, 3:1 and 5:1 ligation protocol at 25 degrees for 3 - 4 hours which has previously worked for me. But after overnight culture of transformed DH5 alpha cells with the ligation mix, no colony was seen on my plates. I equally used the correct antibiotics for the various plasmids, that is Amp = pMAL, Kan = pET28a (+)
I intend to try 4 degrees overnight as well as 22 degrees for 2 hours incubation. Also, I saw online that I could incubate the insert and plasmid at 45 degrees for a few minutes before adding my T4 ligase enzyme. But if there is any other suggestion to optimise this reaction, I would highly appreciate this.
Thanks in advance.
The fact that you are getting no colonies suggests to me that the problem is not with your ligation but somewhere. Doing minor changes in the ratio or the temperature is not going to changes things. It could be that your competent cells are not very good, or that there is a problem with your DNA.
I would start by doing some controls, for example use a tiny amount of each vector (uncut) and be sure you are getting good results (it should be million of colonies and not just a few hundreds). Then you could try and digest the plasmids with 1 enzyme and do a control with and without ligase to be sure your ligase reaction is working well (the number of colonies should go WAY up after ligation). Those will help you start to isolate the problem.
Phil Collins Chidi Tataw how much vector did you use for your ligation reaction? Did you Gel purify your double digested vector, and was purified sample pure? I have previously used a 3.5 kb insert in a 5.6 kb vector. Considering your vector and insert purification as per your message, I would say try 25-30 ng of the vector and insert according to the 3:1 or 5:1 formula, then incubate the ligation mixture at 16*C overnight and do your transformation. One more thing. Did you perform a control set for transformation while doing it with your ligation mixture?
Hello Phil Collins Chidi Tataw , the T4 ligase you are using is supplied...... It is also important to be sure of the purity of the product before ligation. Lastly, try 16 degrees overnight.
Hello Michael J. Benedik , Abir Chakraborty and Robert Adamu Shey thanks for all your contributions. They were really helpful.
I did all the ligation conditions as earlier mentioned in my question. All conditions had colonies and I tried colony PCR analysis for only the 4 degree overnight ligation plates and all the colonies were positively confirmed.
Michael J. Benedik I usually run the double digest with a single digest of each enzyme and undigested plasmid as my controls.
Abir Chakraborty I always use 50 ng of plasmid and always do gel purification after digestion, but no transformation controls.
Robert Adamu Shey I used T4 ligase from Thermo Fisher Scientific and I am certain the problem is not from it.
Thank you all once again.
Phil
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