Protocol Online logo
Top : Forum Archives: : Molecular Biology

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.



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.