How to confirm a 80bp PCr fragment? - (Aug/02/2006 )

I tried to amplify a 80bp fragment, agrose gel showed there was a band near 100 bp in both control and sample. How to confirm it is interest band, now that it is only 80bp, can it be synthesised by company?

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hey Gingerman

Do you know the sequence? Than you could try a digestion (put it on 4% gel) or you can send it away for sequencing.
An other option is that you use primers which are more further away from the area of interest, en when you get a band you perform a nested PCR on the big fragment and see if you get the 80 bp band again.

good-luck!
Maya

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Use PAGE or high-concentrated agarose gel, but controls should be negative to be more sure that it is what you want...Perhaps reamplification from gel band is possible, if the taq accepts the short sequence (I dont know).
Companies also do it, but it is quite expensive, because you need also higher scale of synthetisation.

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3% agarose should do the job with a 50bp molecular weight marker

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Thanks, guys

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1 mM Lithium Borate, pH 8.5 with 3% agarose will provide the resolution necessary to visualize 80 bp.

-Matt

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