lees at lees at
Thu Oct 21 10:45:00 EST 1993

Dear Christine,

Yes, I have been able to activate BspMI digests with  1 mM spermidine on one 
of my best clone (the one with 2 BspMI sites). Higher spermidine conc. at 10mM 
and 100 mM do not work.  However, my 2 other clones with single BspMI site were
not cut under this condition, so I still recommend phiX174 or M13mp18 DNA. 
Can't say if there is a correlation between the number of sites on a particular
piece of DNA and the efficiency of cutting, but it does seems to me that the 
more sites, the better cutting. Probably, binding of the enzyme to one site 
trans-activate cutting at the other?? Does anyone have any idea or wish to
speculate on how this work?
I have previously posted the details of conditions used in my digests to Mike,
but here it is:

1 ug substrate DNA (your DNA)
10 ng phiX174 or M13mp18 DNA
1X NEB buffer 2
BspMI (as much as possible without overloading the glycerol)
I use 3ul (from 1U/ul stock) 
to total vol of 15 ul
incubate 37oC overnight

P/S: An NEB rep told me there is 2000U/ml BspMI available now.

About your other inquiry, John Innes Institute is part (and oldest resident) of
the John Innes Center for Plant Science Research under the AFRC.  It is 
situated in Norwich, south-east of England.  John Innes Center is made up of 
3 large laboratories: the John Innes Institute itself, joined by the Cambridge 
lab and the Sainsbury lab.  A fourth lab soon to be sited here is the Nitrogen 
Fixation lab from U. of Sussex.


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