Problem with TruSeq Custom Amplicon Low Input Libraries

HelenaSC · 05-10-2016, 01:36 AM

Hi all!
We need some help with TruSeq Custom Amplicon Low Input libraries 
We have been processing samples (fresh and FFPE) for a while using this Kit and everything worked fine. Before starting the protocol, we always perform the qPCR to check the quality of the samples and we choose the DNA input as described in the userguide.

Lately, we are experiencing some problems that we don't understand and that seem to be "hazardous", we are not able to see what's going on

…the issue is that we are getting very strange libraries's profiles on the Bioanalyzer 2100 (attached images). Some libraries are not working, others are working ok, and some of them are having some strange peaks before the library (they seem like a kind of "horns") that we can't get ride off, despite purifying the samples again and again….Moreover, it seems that these libraries are carrying something that make the bioanalyzer run not properly, dirty or similar…

Any one has experienced this issue? Any idea of what could it be? The kit is the same that used to work fine some days before, and the controls are working ok randomly!

Thanks a lot in advance!!

Presentación1.jpg

zsmith · 07-22-2016, 09:48 AM

Hi Helana,

Have you cleaned your BioA recently? These sort of profiles can be seen when the pins are dirty.

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05-10-2016, 01:36 AM	#1
HelenaSC Member Location: Spain Join Date: Jun 2013 Posts: 21	Problem with TruSeq Custom Amplicon Low Input Libraries Hi all! We need some help with TruSeq Custom Amplicon Low Input libraries  We have been processing samples (fresh and FFPE) for a while using this Kit and everything worked fine. Before starting the protocol, we always perform the qPCR to check the quality of the samples and we choose the DNA input as described in the userguide. Lately, we are experiencing some problems that we don't understand and that seem to be "hazardous", we are not able to see what's going on …the issue is that we are getting very strange libraries's profiles on the Bioanalyzer 2100 (attached images). Some libraries are not working, others are working ok, and some of them are having some strange peaks before the library (they seem like a kind of "horns") that we can't get ride off, despite purifying the samples again and again….Moreover, it seems that these libraries are carrying something that make the bioanalyzer run not properly, dirty or similar… Any one has experienced this issue? Any idea of what could it be? The kit is the same that used to work fine some days before, and the controls are working ok randomly! Thanks a lot in advance!! Presentación1.jpg

07-22-2016, 09:48 AM	#2
zsmith Junior Member Location: Indiana Join Date: Dec 2010 Posts: 3	Hi Helana, Have you cleaned your BioA recently? These sort of profiles can be seen when the pins are dirty.