restriction reaction on agarose gel slices

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dustman
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restriction reaction on agarose gel slices

Post by dustman » Fri May 25, 2012 2:54 pm

I'm thinking of reducing steps for insert production.

ATM I have following steps:
1. PCR
2. agarose gel with PCR products
3. gel extraction of insert of interest
4. restriction reaction
5. enzyme removal <- I often use KpnI/BamHI and can't go with heat inactivation
6. ligation with backbone and transformation

I know that restriction can be performed on agarose slices but never done it myself. This would save a bit of time and reagents, plus avoid DNA losses in step 5.

Anyone got first-hand experience with this? If 'yes', link to a protocol would be greatly appreciated.

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canalon
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Post by canalon » Wed Jun 06, 2012 1:18 am

Look any DGGE protocol. In my experience it does not save time at all. And neither does it saves n reagents, but buffers are cheap anyway.
Patrick

Science has proof without any certainty. Creationists have certainty without
any proof. (Ashley Montague)

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citroenboom
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Re: restriction reaction on agarose gel slices

Post by citroenboom » Wed Jun 13, 2012 2:46 pm

Why use gel extraction? Often EtBr seems to inhibit ligation. You could use PCR purification kits. Much faster!
Or otherwise go for the good old phenol extraction (Sambrook). Quick, but dirty :)

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