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help-how to extract fluo-labeled DNA from agarose gel


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#1 seagull

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Posted 04 August 2009 - 12:52 AM

from a mixture of 5' end FAM labeled PCR products, i want to have bands between 100-200bp. Not sure how to do.
i am thinking of running the 5'FAM labeled PCR products on agarose gel and then cut and extract bands from the agarose gel using qiagen kit.
but i have two concerns:
1. will fluorescence label be retained to the PCR product after using gel extraction kits? as the gel extraction kit normally do well for unlabled PCR product, but not sure for 5'FAM labeled PCR
2. because 5'FAM labeled PCR product will change mobility as compared to unlabled one, if i look at 100-200bp of unlabled PCR products, how i know what size i should cut for 5'FAM labeled PCR products?

or my stratagy is wrong? should i use PAGE gel? then it is said 5' FAM label may be destroyed by APS. even if run on PAGE, how is the ladder to track the mobility change of 5'FAM labeled PCR product?

looking forward expert to help me......thank you so much!

#2 ph1ll1ps

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Posted 11 August 2009 - 06:36 AM

hi,

I think it's indead difficult to determine what are the 100-200 basepair labeled products.
I don't think FAM will interfer with the gel extratcion kit from Qiagen.

A somewhat wild idea is to use size exclusion columns (millipore), to first loose the fragments <100bp (?YM-10 or ym-30 <100bp in flowtrhough). then use an column with larger pore size to retrieve the 100-200bp (100-200bp in flowtrhough.
but again you don't know how it will react with FAM labeled DNA

good luck




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