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Old 06-04-2020, 03:12 PM   #2
cmbetts
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Location: Bay Area

Join Date: Jun 2012
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Could you explain a little more about you're intending to accomplish? As I understand it, you're performing paired end sequencing on a MiSeq. You then take the spent flowcell, and perform the described molecular biology steps with the desired result being dsDNA anchored to the flowcell in the same orientation as at the end of read2. Is that correct?

If so, I don't think that the Read1Cy3 oligo is a valid readout. The structure of your desired product has the sequence identical to the read 1 primer and it's reverse compliment annealed to it from the regenerated strand. I wouldn't expect a Read 1 oligo to be informative here because you'd have to displace your new strand to hybridize to it. Have you considered labeling the Read2 extension primer? Or maybe use a small portion of labeled nucleotide in your extension mix? This may not be compatible with your final downstream use, but could be used until you are confident in your protocol where it can be discarded. This is all assuming that you have access to a relatively plentiful supply of discard flowcells from other users at your institution that were going to throw them away anyhow.
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