Problems digesting with BclI and SspI - Any help is apriciated (Jan/18/2005 )

I am trying to do a double digestion with BclI and SspI both enzymes from NEB.

I am having a problem, i need to cut a plamid that is 8.8kb and release a 700bp fragment.

BclI needs to be digested at 50C without an inactivation step. SspI can cause star activity.

I have digested with either enzyme first and played with the amount of SspI enzyme to use. I am seeing my plasmid linearize with the first enzyme digestion, (i clean up my reaction with a quiagen kit) then digest with second enzyme. The problem is that i am not seeing the 700bp fragment after digestion. I need to use 5ug of DNA for a cloning reaction down the line. I am interestred in the 8.1Kb fragment not the 700bp.

Have any of you used these enzymes together or separate? Any help on this will help.

-izzy

Pay attention to the ratio of the 700 bp band to the plasmid.

A 700 bp band from a 3kb plasmid is much more visible than a 700 bp band from an 8 kb plasmid, if you used the same amount (ug) of DNA for digestion.

1 ug of 3KB plasmid has more plasmid molecules than 1 ug of 8kb plasmid.

Try bumping up the amount of plasmid for digestion.

Since you said that both enzymes linearize your plasmid, then I dont think there is an issue with the activity of the enzyme.