I want to insert a DNA fragment of 16oobp into the pTRE2hyg vector with the site of Cla1 & sal1. It seems that these two Enzymes are not so high efficient to digest with.Would anyone give me some suggestions about which one should be used firstly to digest the vector?

Thanks!

If no compatible buffer for both enzyme, I think the only way is to do it one by one. First RE --> Purification --> Second RE

According to NEB's double digest finder

http://www.neb.com/nebecomm/DoubleDigestCalculator.asp

You could do a double digest with these enzymes (if you use NEB enzymes use buffer 3 +BSA at 37 C) but the ClaI will only work at 50% efficiency, so just add twice as many units of ClaI than SalI.

Sal I should not be a problem, but Cla I can be depending on the sequence context. It can be methylated. For this reason, I would certainly not try doing a double digest but instead do Cla I first and test a little on a mini gel after about 60mins to check that it was complete. If it was then do a GeneClean or other purification and carry out the Sal I digest.