restriction enzyme specificity - (Nov/30/2005 )

I have been trying to place an insert into a pCMV.Sport6 vector. My latest pair of restricition enzymes are BamHI and XhoI. I keep getting two bands - both of which add up to the size of my insert. Although there are no specific restriction sites for these two enzymes within my insert there is a ggagcc - at about the right place in the sequence. Could BamHI be targetting this sequence?

-freyja-

Could be. Do each digest separatly and see what you get. That will pinpoint the problem enzyme.

If you want to be paranoid, set up a mock digestion (everthing except RE).

Are you adding BSA to your digest??

Try reducing the amount of RE and see if that helps.. or reduce digest time.

-pBluescript-

This sounds like classic star activity, to which Bam HI is susceptible. NEB defines star activity nicely (from their Bam HI star activity FAQ:

QUOTE

It has been demonstrated that under extreme non-standard conditions, restriction endonucleases are capable of cleaving sequences which are similar but not identical to their defined recognition sequence. This altered or relaxed specificity has been termed "star" activity. It has been suggested that star activity may be a general property of restriction endonucleases and that any restriction endonuclease can be made to cleave noncanonical sites under certain extreme conditions.

The FAQ also lists several conditions under which Bam HI might exhibit star activity:

QUOTE

Conditions that Contribute to Star Activity

High glycerol concentration [> 5% v/v]
High units to µg of DNA ratio [Varies with each enzyme, usually >100 units/µg]
Low ionic strength [< 25 mM]
High pH [> pH 8.0]
Presence of organic solvents [DMSO, ethanol, ethylene glycol, dimethylacetamide, dimethylformamide, sulphalane]
Substitution of Mg++ with other divalent cations [Mn++, Cu++, Co++, Zn++]

Or, perhaps there's an error in your sequence, and the Bam HI site is really there. Did you sequence it yourself?

-HomeBrew-

Thx - I did let the rxn go for 3 hrs and buffer was not entirely specific to these enzymes. Have not sequenced the insert ourselves yet...

-freyja-

hi
i doubt about a star activity on 3hours only. Can it be a non digested and single digested bands.?

-fred_33-

I've never thought of star activity as a time-dependant thing, fred -- if the conditions are correct for star activity, the enzyme exhibits aberrant digestion, right from the start. No?

-HomeBrew-

hi
i though it was part of an equilibrium. At beginning the enzymes can "meet" targets easyly so star does not occurs... But this may be totally false. I try a search on that point and keep post informed

-fred_33-

Hmmm... Good point. It might be equilibrium driven -- now I'm not sure...

-HomeBrew-