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double digestion

double digestion - DNA Techniques

double digestion - Post questions and discuss DNA techniques and protocols such as DNA extraction, PCR, and the study of DNA-binding proteins.


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  #1  
Old 08-22-2006, 06:39 AM
Pipette Filler
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Default double digestion



Hello,
I am working on plant DNA and have to perform a double digestion with two different enzymes. One has a high salt buffer an incubation temp.37 degrees and the other has a low salt buffer with incubation temp 65 degrees. how should I go about it
thanks
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Old 08-22-2006, 03:20 PM
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Default Re: double digestion

Hey Ress!

Welcome to the forum!
Find out which one has the least salt buffer... you can then digest the DNA first for this restriction enzyme and then can optimize for the second enzyme which requires the most salt.

OR check NEB company catalogue or website. they have a chart for double digests. see if you can digest with them both, what percentage both will be active and also which buffer is best for both of them.

cheers!
Kiki
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Old 08-23-2006, 08:29 AM
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Default Re: double digestion

Thanks kiki
The problem is the high salt enzyme is the rare cutter(6 base) and I have to use it before the 4 base cutter. I would have to digest the DNA then ethanol ppt it then digets it with the second enzyme. It is a lengthy process and I wanted to avoid it.
I am from India and ordering the enzymes from NEB is quite costlier, can you suggest something.
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Old 08-23-2006, 04:51 PM
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Default Re: double digestion

Hey Ress!

NEB lists a double digest chart I can post the link.

Double Digest Chart NEB

Also dont worry NEB also lists the recipe of the buffers used in the digests I am sure you could easily set them up

Buffer compositions

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