Restriction enzyme digest

HIRANYA ROYCHOWDHURY hroychow at NMSU.EDU
Wed Apr 17 17:48:33 EST 1996


On Wed, 17 Apr 1996, sujoy ghosh wrote:

> I am planning on doing a double digest of a PCR product and a vector with
> BglII and SpeI.  Knowing that some REs are notorious, does anyone have any
> advice on the reaction conditions for this digest? I am using Gibco's
> enzymes. I am enquiring specially about time, sequence of enzyme addition
> and things like that.
> Sujoy (sujoy_ghosh at glaxo.com)
> 
> 
Hi  Sujoy,
	I routinely use the Pharmacia buffer system for most cuts and
BglII and SpeI do work together in that. The buffer, at 1x, would have
10mM MgOAc, 50mM KOAc buffered with 10mM Tris-acetate at pH 7.5. However,
BglII has a lower activity in it (about 50% to 60%) and so I would add it 
first and digest for 1 to 2 h before adding SpeI. 

Hope this helps,

HSRC.

			>>>>>>>>>>>>>>>>>>>>>>>>>>>
			  Hiranya S. Roychowdhury
   			  Plant Genetic Engineering Lab.
			  Box 3GL, NM State Univ.
			  Las Cruces, NM 88003
			  Phone: (505) 646-5785
			  hroychow at nmsu.edu
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