bioanalyzer image

polystone · 03-29-2010, 11:52 AM

I am preparing the library for single end RNA-seq following the illumina protocol. However, by agilent analysis for library, the two smear bands occur at 200bp and 500 bp. I do not know why the 500 bp band is visible. Who has the same experience? Anyone's idea for this result?

peromhc · 03-30-2010, 11:30 AM

What stage of the library prep are you at?? Is this the final purified library? What size bands are you selection?

polystone · 03-30-2010, 12:21 PM

Quote:

Originally Posted by peromhc

What stage of the library prep are you at?? Is this the final purified library? What size bands are you selection?

It is purified library for subsequent cluster generation in mRNA-seq. This library is generated by PCR enrichment during which the gel-seclection size at 200 bp is used as template. The PCR cycle is 15. Thus, in theory and according to illumina protocol of sample preparation for single end RNA-seq, the band shoud be only at around 200 bp, and the 500 bp band should not occur. I do not know why 500 bp band is yielded? I need 200 bp band.

LMcSeq · 03-30-2010, 07:44 PM

What about running another gel and selecting only the size you want?

peromhc · 03-31-2010, 07:40 AM

Poly, I would follow LMc's advice and do another round of size selection- it's hard to know what that band might be, but Im pretty sure you dont want it!

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03-30-2010, 11:30 AM	#2
peromhc Senior Member Location: Durham, NH Join Date: Sep 2009 Posts: 108	What stage of the library prep are you at?? Is this the final purified library? What size bands are you selection?

03-30-2010, 07:44 PM	#4
LMcSeq Member Location: Maryland--we have a HiSeq too! Join Date: Feb 2009 Posts: 33	What about running another gel and selecting only the size you want?

03-31-2010, 07:40 AM	#5
peromhc Senior Member Location: Durham, NH Join Date: Sep 2009 Posts: 108	Poly, I would follow LMc's advice and do another round of size selection- it's hard to know what that band might be, but Im pretty sure you dont want it!