ExoSap purified PCR product compatible for Illumina sequencing?

wingtec · 10-29-2014, 07:01 AM

Hi All,

Does anyone have experience with purification of PCR amplicons using ExoSap Endonuclease treatment? I wonder if this procedure is compatible with MiSeq sequencing.

Best,

Wing

cmbetts · 10-29-2014, 12:08 PM

I haven't tried it, but I'd be concerned that still having all of your PCR buffer around would interfere with the denaturation and flowcell annealing steps of loading the instrument, which seem to be the most finicky steps in my experience.

wingtec · 10-30-2014, 11:55 AM

Thanks, CMBETT.

For me a bigger concern is wether this will mess up the denaturation step, especially in the case where the ExoSap reaction does not get high enough dilution based on quantitation. Quantitation after the purification is also a concern, because of the introduction of the proteins.

Best,

Wing

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10-29-2014, 07:01 AM	#1
wingtec Member Location: Charlottesville, VA Join Date: Apr 2010 Posts: 34	ExoSap purified PCR product compatible for Illumina sequencing? Hi All, Does anyone have experience with purification of PCR amplicons using ExoSap Endonuclease treatment? I wonder if this procedure is compatible with MiSeq sequencing. Best, Wing

10-29-2014, 12:08 PM	#2
cmbetts Senior Member Location: Bay Area Join Date: Jun 2012 Posts: 118	I haven't tried it, but I'd be concerned that still having all of your PCR buffer around would interfere with the denaturation and flowcell annealing steps of loading the instrument, which seem to be the most finicky steps in my experience.

10-30-2014, 11:55 AM	#3
wingtec Member Location: Charlottesville, VA Join Date: Apr 2010 Posts: 34	Thanks, CMBETT. For me a bigger concern is wether this will mess up the denaturation step, especially in the case where the ExoSap reaction does not get high enough dilution based on quantitation. Quantitation after the purification is also a concern, because of the introduction of the proteins. Best, Wing