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Which digestion enzyme?


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#1 Tired

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Posted 17 September 2009 - 10:32 AM

Hi Bioforumers,

I did a PCR of my DNA fragment and now would like to ligate it into a pIB/V5-His vector. I assume that I should do a digestion of the vector and my DNA first to create "sticky ends".

My question is, which digestion enzyme should i use? Should I use the restriction enzymes that I used for my PCR primers (BTW they were Xho I and Sac II) or do I just use standard EcoRI?

Is there a specific protocol for this digestion? If so, could someone post this protocol?

Thank you very much for any suggestions

:)

#2 leelee

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Posted 17 September 2009 - 06:48 PM

Do you have someone in the lab to help you with this? Perhaps speak to the person who designed the primers for you- as they will have chosen SacII and XhoI for a reason. It seems that you don't have very much background knowledge about how ligation works- my advice would be to go and talk to someone and read up about the procedure before you do anything else.

If your PCR fragment has an XhoI site on one end and a SacII on the other (that is how I am understanding your post??), then you will need to digest this product with those enzymes- and your vector also. You can't do an EcoRI digest for your vector as the sticky ends will not be compatible with your PCR fragment's XhoI and SacII ends, and you can't do EcoRI digest for your PCR product, as you don't have EcoRI sites added to the ends.

With regard to RE digest protocols- go to the manufacturer's website and they will have optimal conditions for each of the enzymes they supply, this should also be on the product insert that comes with each of the enzymes.
(for example, for NEB, http://www.neb.com/n...ategory1.asp?#2 )




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