Hi all,
I am new with cloning and I am trying to carry out a ligation with three elements (two inserts and the vector). All of them must be cut with restriction enzymes and run on the gel before the ligation reaction. I don't know if it is better to run all the digestion reaction on the gel and cut the bands (corresponding to the fragments digested ), clean them up and carry out the ligation or run just a part of the reaction on the gel and leave some volume of each of those for the ligation. I was told that the first choice would be better in order to maximize possibility that the ligation with all the three elements works..but it is not clear to me why.
Thank you very much
In my experience, it is best to purify and quantify each component individually then perform the ligations. This allows better control of the insert/backbone ratios and permits easier optimization. For this reason, it makes sense to run out all of the digestion reaction.
The only reason to run out a small amount of the digestion reaction is to confirm that it has gone to completion.
There are certain circumstances where a gel-extraction is not necessary to purify a component needed for part of a cloning scheme (e.g. a backbone cut at a single site, or at two very nearby sited can be purified over a column, which will not bind very small ss- or dsDNA oligonucleotides).
You can always use a small part of your digestion results to check in a gel if the bands are present. After checking the bands, you can either run the whole sample in a gel, cut it out and purify DNA or use your remaining samples for ligation.
However, if you gel purify your samples, you remove any non-specific bands or any other non-target DNA sequences. So the purity of your sample is higher compared to a sample that has not been run on a gel.
It is true that you can lose upto 20% of your sample DNA during gel purification, but for ligation and cloning, the purer the components, the better.
For cloning, Different steps (digestion and ligation) should be checked only by running a gel with whole sample. By this process you will get all the gene parts of your interest accurately. And by that time you should always run respective 'controls'.
Hi,
Hope you are going to clone all three bands; two insert and the vector backbone. And it's a mixture of all three bands resulted after restriction digestion.
If you want to clone three band separately you must load all the mixture and separate fragments through gel( it possible only if you have bands with three different sizes). Then cut each band separately and gel purify. Use those purified fragments for the ligation mixture.
If you have digested the vector with different restriction enzymes I think you can clone without fragment purification but I don't sure about that. Because cloning means ligation of fragments is a random process which can yield different ligated products and there is a possibility of religation of the fragments you already digested. So it won't be a good method anyhow. it will cause more problems in future. But It's depend on your requirement of cloning which I don't sure.
Hope you got something. It's not easy to answer without knowing what actually you did and what you are going to do. Wish you a Good luck in cloning.
Hi
After restriction you have to purify your fragments anyway to remoove restrictases and small parts of inserts (If you cutted PCR product to have sticky ends, for example). It possible to use spetial Kits or run fragments on gel. Otherwise your ligation may be uneffective.