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Digest with AarI and Pml I

restriction digest cloning

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#1 Shiny

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Posted 04 June 2012 - 08:26 AM

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

#2 phage434

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Posted 04 June 2012 - 02:06 PM

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.





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