DNA binding by RE causes bigger band than substrate in gel? - Restriction digest of 500bp frag. with BsaXI (Apr/19/2005 )
I am trying to a digest a 500bp fragment (for development of a PCR-RFLP protocol for ID of diptera...don't worry!) and one of the enzymes I need to use is BsaX I (New England Biolabs).
Unlike digests with the other enzymes I've used, where the enzyme either digests the fragment (smaller bands result) or doesn't digest the fragment (same fragment remains when run on gel), this enzyme seems to cause ALL fragments it's incubated with to run approximately double the size ofthe original fragment! I have no idea why and would REALLY love some suggestions as my RE knowledge is severely limited.
On the cert of analysis it says that the enzyme can cause "DNA binding" if incubated greater than 2 units in a 16 hr incubation. Could this binding capacity of the enzyme cause the strange large fragments to be observed???
PLEASE PLEASE HELP ME!!
I read the write-up for BsaX I and I guess that is what you're seeing.
I'm wondering, why such an ugly enzyme as BsaX I? can't you use any other enzyme?
you can call NEB and ask them about it.
[COLOR=purple][quote=leigh,Apr 20 2005, 04:46 PM]
I am trying to develop a PCR-RFLP assay to differentiate blowflies of forensic importance, and unfortunately for the species I am trying to differentiate their sequences are almost identical due to their close genetic proximity - hence why only this enzyme should (in theory!) work to cut them differentially. Does BsaX I have a bad rep in the RE world??!!
I'm sorry, that's not what I meant about "ugly". And I understand now why you have to use BsaX I. It's just that I looked at its recognition sequence and you can imagine my reaction.
I would really try to call NEB and ask them what they mean by "DNA binding." That may be what your seeing.
so if I got it right your problem is to seperate bands on the gel after the RFLP-PCR and digest with the enzyme, right?
If so and if your enzyme sticks to your DNA you can add SDS (0,1-1% v/v) to your DNA Fragments you put on a gel as this might disrupt the enzyme binding to the fragments.
I did this with the homing endonucleases P-SceI and I-CeuI as these enzymes also tend to bind DNA.