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Re: polysaccharides in RNApreps from plants


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#1 anonymous

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Posted 01 March 2001 - 10:00 PM

From: Vertino P.
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Category: General method

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Dear netter,

I have amplified my pcr products with primers which have a restriction site introducedinside 4 extra nucleotides, and have tried many times to clone my pcr products (afterdigesting with enzymes) into a plasmid cut with the same enzymes. it simply did not work.Can anyone give me a clue?

Thanks in advance.

Vertino


#2 anonymous

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Posted 02 March 2001 - 10:00 PM

From: Samir
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Category: General method

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I think the posible cause may be the enzyme you used to cut your pcr products doesn'twork well with 4 extra bases outside it's cutting site. you may have to add 2 or 3 morebases to your primer or TA clone your pcr first then subclone into your expression vectoror any other vector.

Good luck

Samir


#3 anonymous

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Posted 07 March 2001 - 10:00 PM

From: craig
email: craig.mcandrew@kcl.ac.uk
Category: DNA-Related

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It's true that it is possible that your enzyme does not cut efficiently with a fourbase overhang, you can check in the back of the N.E.B. catalogue. It may also beworthwhile to digest your PCR products with a vast excess of enzyme, the units of activityrefer to ug's of lambda DNA with is something like 20kb.





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