Digest with AarI and Pml I - (Jun/04/2012 )

Hello everyone,

I have a very weird problem and was wondering if anyone has ever encountered a similar problem. I want to digest plasmid DNA using Pml I { Isoschizomer PmaCI (NEB) or Eco72I (Fermentas) } and AarI. I cannot digest the DNA with second enzyme, irrespective what ever order I start with. I do a sequential digest rather than a double digest. I change the buffer by following qaigen PCR purification. I have tried to linearize with PmlI but then AarI would not digest and vice versa. The sites are there as single digest works fine. I am completely confuse as to what is the problem with the digestion!!

Band sizes

Vector size ~6.9kb, double digest 6422bp, 447bp
Insert size - 623bp, double digest 448bp, 174bp

Please help

thanks

Shiny

The sites may be too close to one another, or even overlap. Most enzymes will not cut near the ends of linear DNA fragments, so this can be a problem.