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Old 10-30-2012, 05:39 AM   #3
pmiguel
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Location: Purdue University, West Lafayette, Indiana

Join Date: Aug 2008
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Although the LRPCR approach would be ideal, in many cases it isn't realistic. NexteraXT should be able to deal with amplicons in the 400-900 bp range okay.

But I think you are right, the 0.6x AMPure will remove most of the products you want. If possible, do a high sensitivity chip after AMPure to see what is there. Subtract 130bp from the sizes you see -- that will be your insert sizes. If acceptable, you should be fine. Whether you use normalization or not.

If you don't normalize, you will need to use the NaOH denaturation procedure, rather than the heat denaturation (which is for ssDNA and may not work at all for dsDNA).

BTW, even if you do normalize, qPCR is still an option for QC.

--
Phillip
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