Can't see my library on a gel

Mollycule · 09-06-2012, 06:37 PM

Hi everyone, sequencing newbie here!

I'm finishing up my first Illumina RNA-seq library prep, and my protocol suggests running my library out on a gel to check for a smear of the appropriate size (~300 bp). But when I do this, I don't see my library on the gel--even after 18 cycles of amplification, and even with a sensitive stain like SYBR Gold. Well, I do see faint adapter dimers, but I suspect these can be removed with Ampure XP cleanup. Does this mean my library yield is too low to proceed with sequencing? What is an acceptable yield range, and how do you guys do your library QC?

Any input is appreciated. Thanks!

jinpingzhang · 09-06-2012, 09:23 PM

Hi,
I am not sure Illumina RNA seq library length and concentration. but i am sure the SOLiD RNA library some information. After your PCR and purification process, you will get several ng library, use Agilent High Sensitive DNA Chip check them.

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09-06-2012, 06:37 PM	#1
Mollycule Junior Member Location: Ithaca, NY Join Date: Sep 2012 Posts: 1	Can't see my library on a gel Hi everyone, sequencing newbie here! I'm finishing up my first Illumina RNA-seq library prep, and my protocol suggests running my library out on a gel to check for a smear of the appropriate size (~300 bp). But when I do this, I don't see my library on the gel--even after 18 cycles of amplification, and even with a sensitive stain like SYBR Gold. Well, I do see faint adapter dimers, but I suspect these can be removed with Ampure XP cleanup. Does this mean my library yield is too low to proceed with sequencing? What is an acceptable yield range, and how do you guys do your library QC? Any input is appreciated. Thanks!

09-06-2012, 09:23 PM	#2
jinpingzhang Member Location: Houston Join Date: Mar 2012 Posts: 10	Hi, I am not sure Illumina RNA seq library length and concentration. but i am sure the SOLiD RNA library some information. After your PCR and purification process, you will get several ng library, use Agilent High Sensitive DNA Chip check them.