Problems with restriction enzymes - (May/31/2005 )

I have been battling with restriction enzymes for a while now. I am attempting to free an insert from TOPO pCR 2.1 using an engineered Bbs I site and either a BamH I or Xho I site found in the vector, depending on the orientation of my insert. All enzymes are supposed to efficiently cut in NEB 2 (+BSA). When used separately, I am able to linearize my construct will all 3 enzymes. However, I am unable to do double digestions with Bbs I/BamH I or Bbs I/Xho I (I cannot see my insert fragment). I have tried doing serial digestions (PCR purification column used between digestions to get rid of enzyme), starting with either BamH I/Xho I or Bbs I, to no avail. I was wondering if anyone has any suggestions. I was thinking that my starting DNA may contain something that is inhibiting the reaction, but the fact that I am able to cut the DNA with each individual enzyme makes it seem like this is not the problem. Thanks for any guidance.

-Jesse

Hi Jesse, I checked the catalog book from NEB, looks like BamHI need BamHI buffer to work efficiently. According to their double digestion instruction, I think you should try digest with BbsI first (NEB 2 without BSA), then add certain amount of NaCL to bring the salt concentration to 150mM while digest with BamHI. For BbsI and XhoI, the salt concentration does not to be changed, just add BSA and adjust the volume of the reaction. Hope this is helpful.

xiaoyang12

Hi Jesse,

How big is your insert? If it's too short, it may not easy to be seen on gel.

Have you tried sequential cut? You can cut first with the enzyme which requires low salt buffer and then add more salt to the reaction to the concentration required by the 2nd enzyme. Alternative, after the 1st cut, purify the plasmid and cut with the 2nd enzyme.

Hope that helps.

Paulina

you can try using two enzymes plus buffer with the lower NaCl concentration... them you incubate overnight to 37°C.