MiSeq sample volume, recover leftover library after run?

JBKri · 10-22-2015, 08:38 AM

A MiSeq run needs 600 ul of denatured library. However, about 415 ul of library remains after the run has completed (measured after a v3-600 run). Would it be possible to load a smaller volume? If used in combination with the NextSeq denaturation protocol, a smaller volume would enable a higher cluster density from low-yield samples. This would only work if the sipper goes deep enough into the tube containing the library.

Another (crazy?) thought is to recover the leftover, denatured library, and re-use it in another run if needed, unless there is some sort of backflow or mixing from other wells back into the sample well. Again this only makes sense for low-yield, precious libraries. Any thoughts?

An additional thought; PCR-free library protocols are generally (always?) less biased, but require large amounts of input DNA. The approach with NextSeq denaturation, and possibly less dilution before loading into the cassette, could potentially generate usable libraries from smaller starting amounts of DNA. That seems like a better solution than adding a PCR enrichment step as I discussed here.

Jon

kcchan · 10-22-2015, 11:23 AM

Almost all of that volume is water from the template rinse.

JBKri · 10-22-2015, 11:51 AM

Quote:

Originally Posted by kcchan

Almost all of that volume is water from the template rinse.

Ah, I see. So my idea wouldn't work. Thanks!

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10-22-2015, 08:38 AM	#1
JBKri Member Location: Heraklion, Greece Join Date: Jan 2014 Posts: 79	MiSeq sample volume, recover leftover library after run? A MiSeq run needs 600 ul of denatured library. However, about 415 ul of library remains after the run has completed (measured after a v3-600 run). Would it be possible to load a smaller volume? If used in combination with the NextSeq denaturation protocol, a smaller volume would enable a higher cluster density from low-yield samples. This would only work if the sipper goes deep enough into the tube containing the library. Another (crazy?) thought is to recover the leftover, denatured library, and re-use it in another run if needed, unless there is some sort of backflow or mixing from other wells back into the sample well. Again this only makes sense for low-yield, precious libraries. Any thoughts? An additional thought; PCR-free library protocols are generally (always?) less biased, but require large amounts of input DNA. The approach with NextSeq denaturation, and possibly less dilution before loading into the cassette, could potentially generate usable libraries from smaller starting amounts of DNA. That seems like a better solution than adding a PCR enrichment step as I discussed here. Jon Last edited by JBKri; 10-22-2015 at 09:38 AM.

10-22-2015, 11:23 AM	#2
kcchan Senior Member Location: USA Join Date: Jul 2012 Posts: 184	Almost all of that volume is water from the template rinse.