ligation help - (Sep/17/2004 )
I want to ligate a 500bp DNA into the vector pET9-a. I had already double digested the vector plasmid and the insert (500bp DNA). The digested plasmid was also purified by extraction from the agarose gel. The ligase I use is T4 ligase from Fermentas. I follow the protocol they give us.After ligation is done, I run a gel but can't see anything. Since I don't know the concentration of the plasmid and the insert. I make 20 microliter sample with 8 microliter plasmid and 10microliter insert.
The complete protocol is below:
8 microliter vector
10 microliter insert DNA
2 microliter 10X T4 ligation buffer
0.4 microliter T4 ligase.
Incubate at 22 degree for 1 hour;
Inactive T4 by heating the reaction at 65 degree for 10 min.
Bs: I load 10 microliter of the mixture while I run the gel?
Can anybody tell me what I can do ? Thank you very much!!
Well, if you don't see anything on your gel, you either have insufficient amount of DNA to see, or forgot to add ethidium bromide to the gel. You write the volumes of your vector and insert but what is their concentration? Usually, you cannot see more than <100 ng of DNA on an agarose gel, maybe that's the reason?
Do you know how much plasmid you initially added for digestion? You should have an idea how much plasmid and insert are used in the ligation reaction. After the ligation, load 4 ul onto a gel, you should be able to see something on the gel. If ligation is good, you will see circular plasmid (two bands). I agree with biotech, those he mentioned are potential problems.
Firstly thank you very much for your reply!
I use fluorometer to detect the concentration of purified plasmid. It is very low only 15-20ng/ul. The digested insert isn't purified after the double digestion since I loss almost all the DNA while purification. Do you think it is necessary to purify the insert firstly before ligation?
Can I use the ligated mixture for transformation even though I can't see the bands in the gel?
Hope to see your reply soon!
Thanks a lot!
I got the same problem today.
I am running the plasmid after ligation and there is nothing on the gel.
I have above 20 ng, and I can see this concentration - I have done control of that.
A few things:
- You want more insert than vector, so swap your volumes around.
- You must do something to purify the insert -- how are you removing the restriction enzymes used to digest it? What restriction enzymes are you using?
- Just because you can't see the ligation product on a gel doesn't mean your transformation will fail. What happens when you transform?
Don't look, just transform. Running ligations on a gel is difficult, and difficult to interpret. Usually the concentrations are low, the volumes are low, and the results ambiguous. You're better off just transforming the results.
Agreed. I never look at a ligation mix on a gel -- the proof of successful ligation is a successful transformation, not a band on a gel.
I sent the copy to Invitrogen technical support yesterday:
I have a problem with ligation.
I tracked the experiment on agarose gels and:
- I have the right digestion
- After gel purification of both insert and vector, i run the solution on the gel - and there are nice crisp bands in the right position...
- I add gel purified insert and vector to ligase buffer, water and 0.3 unit of ligase [sticky ends]. And no something weird happens. If I decide to run this ligation reaction on gel - it appears that there is no DNA. [!] What is even more interesting in one case I found a bit diminished band, in two other cases not. in the aliquots from earlier ligations stored in -20C over 24 hours there was no DNA as well.
Do you have any idea what could be going on?
If it is possible, could you give me the protocol for ligation downstream [from restriction digestion] using your T4 ligase which you think is most suitable for your buffer and ligase?
So the problem is, that I am running gel purified fragments - and they appear. I add the same DNA to the ligation reaction [about 20-30 ng] - and they are inexistant. 5'-3' ligase activity? WTH??? Any ideas what I can be doing wrong? Why running ligation on gels is harD? How can i increase the sensitivity of gels? Making them thinner?
And btw. Maybe some knows:
I am adding very small inserts: 100bp, 400bp, and 500bp, what should be my approach [some references]? I am using invitrogen T4, and protocol which is supplied with the tube.
Maybe there is some nuclease in your purification reactions... especially if you can see the purified DNA right after purification but not 24h later from the same sample??? ligate and transform quickly??? clean up your gel box and use new razors etc???