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enzymatic digestion


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#1 helene

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Posted 05 April 2002 - 11:25 PM

hi! I'm trying to digest a RTPCR product with 2 restriction enzyme but I'don't have any good results : on 2% agarose gel, I can see some expected bands but not all of this (2100,647,347,268,247,180 pdb). Furthemore the fluorescence is weak despite of a good coloration in BE and of use of a great amount of RTPCR product. Before the digestion, I purify the product with a Quiakit pcr purification kit. The time of incubation is 2h30 (37°C).

 Do you have a solution for this problem?

     Thank you!

     Hélène


#2 franck

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Posted 09 April 2002 - 07:06 AM

Are you sure of your kit? Is it really a good one, or is it really necessayr, because i have the feeling that kit catches a part of your sample.

#3 AronD

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Posted 09 April 2002 - 10:50 AM

helene,

Have you tried using BSE in the RE buffer?  I know that it sometimes helps if the enzymes aren't cutting.  The other thing to try is spermidine, but I would try the BSE first.  You may also want to try incubating overnight to make sure that the cDNA is cutting fully.  Good luck!


#4 AronD

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Posted 11 April 2002 - 05:15 AM

Helene,

Seems I have cow diseases on the mind.  I should have written BSA (bovine serum albumin) not BSE.  Sorry about the confusion.

Aron





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