how to check if restriction digestion has happened - (Oct/18/2007 )
Hi,
I am trying to digest a vector and a PCR product with BamHI and SalI. Then I can ligate the PCR product into the vector. But how do I tell if the digestion has happened or not. I do 37 C, overnight incubation with the restriction enzymes. The undigested PCR product and vector will be only a few bases bigger than the digested ones, so I don't think there will be any difference ona gel between undigested and digested DNA. So how can I tell if restriction digestion has happened?
Run the undigested vector along the digested vector and you will see a difference in the way they run (coiled DNA vs linear DNA).
For PCR product, Its not possible to know if its digested or not. Just pray that it got digested.
Yes indeed. Welcome to the black arts of molecular biology.
You will find that practitioners of this dark art have more faith and prayer then many religious followers. You will only know if the PCR product is okay if the ligation works. if it doesn't, then it is a matter of slowly eliminating possible suspects. Not so. Simply cut the product with both enzymes. Then, heat kill and/or purify the DNA. Then, ligate the DNA. Ideally this will form double length circular fragments or longer even-length circular or linear fragments. Heat kill the ligase. Digest with each of the enzymes and with both. Run the undigested ligation product and each of those three digestions on a gel. You should see primarily long fragments in the undigested ligation product. You should see primarily double length fragments in each of the single enzyme digested samples. You should see primarily single length fragments in the two-enzyme digested ligation product. These tests directly tell you whether the DNA was correctly digested and was then able to ligate, which is what you care about. You can easily tell which enzyme is having trouble, or which ends are not easy to ligate. No black art. Engineering.
what about a third enzyme that cuts only the insert (though not always needed, if you know the sizes that it should be). if you do a separate digestion with Bam1 and X or Sal1 and X a specific length fragment would drop out- voila you have your insert!. Of course running the undigested vector is probably the easiest and fastest way. This way you can get information on orientation too. Just an idea
Good luck
as always
Lost in the Lab
To make sure you are cutting your PCR fragment you can blunt end ligate it some vector (preferably one that has blue/white screening so its makes it easy for selection) that way you will know if its been cut or not and have an unlimited supply of your insert. Its a bit extra work but it can eliminate you worrying about your PCR product not being cut.
Or you could clone it into a TOPO-vector and cut it out afterwards, maximum 1day more work...
Since this is the matter of simple ligation, try not to complicate too much/at all. The PCR product should easily be cut with both enzymes, since it is a linear fragment (providing you have added enough bases on each side of the restriction site). If you do encounter problems, it will most likely be at the stage of vector linearization. Enzymes do not cut supercoiled DNA as efficiently as they do linear. As Scolix says, run both (the cut and he uncut) vectors on a gel (best if low percentage - 0,8%). You will see the undigested one run faster. You just need to purify the digested one and along with your ligation also set up a back-ligation experiment. If the number of colonies on your back-ligation plate is lower than on the ligation plate, you will for sure have positive clones.