how many bases are needed upstream of restriction site? - (Dec/03/2005 )
We are trying to digest PCR product and clone into our vector - absolutely no success yet. We are using SphI and XmaI and have 6 bases upstream of the RE site (based on some guidelines from the NEB site). Is this sufficient? Any suggestions?
Thanks a lot
What do you exaclty mean with no succes?
Do you, after ligation and transformation, get no colonies or just empty colonies (vector self ligations)? What are your results for the negative control reaction? Have you checked concentrations of the cut vector and the cut PCR product? What were the molar ratio's you tested for the ligation? ARe your restriction enzymes methylation sensitive (to make sure your vector is cut twice). What's the distance between the restriction sites on your vector (can you distinguish between single cut and double cut vector if you run it on a gel)?
Anyways, 6 bases doesn't seem enough for me. Add 4-10 more, and you might be more succesfull. Or otherwise, do your PCR, clone it into a TOPO vector (blunt topo or Topo TA depending on the enzyme your using in your PCR), cut your fragment out of the topo-vector, gel-purify and then ligate.
At first, have you done purification of PCR products before digestion?
And, for the question about required number of bases - see here, for example.
go to the back of the New England Biolabs Catalogue. There is a host of excellent information and information on the activity of restriction enzymes. It will tell you how many nucleotides a restriction enzyme needs and how efficient it is at digesting. In some cases, adding 6 nucleotides doesn't do much, you may only get 20% digestion.
oops...you have already gone to the NEB catalogue!! Sorry...I have added an sphI site to a primer and digested the PCR product successfully. I don't know about the xmaI one.
6 bases are required generally.
But if you don't succeed in the next exp, i ould suggest to do a topoTA cloning (or pGEMT depends on your purpose). You'll therefore have a pcr fragment in vector and no need to brainstorm about the number of bases...