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Old 04-24-2013, 12:13 AM   #3
Location: earth

Join Date: Mar 2009
Posts: 68

It depends on what you are trying to do. If you want to retain strandedness, then I'd suggest tagging individual molecules, fragmenting, sequence, reassemble, then get on with the analysis. I haven't tested this.

If you are just interested in the first 200 bp (assuming fusion here), fragment select for 200 bp, ligate on only the P1 adapter, then get going. I haven't tested this.

Or you could redesign the primers so they only work for 200 or 400bp chemistries. I usually go this route. The question then becomes which of these sequences do you want to miss with the new PCR design? Do you have a good bioinformatician?

The real limiter here is that the emPCR reaction limits the length of the input fragment. See if you can make a fragment that fits within the specifications of the kit you are using while still answering the question you are asking.
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