Shoulders/humps seen in DNA library

mollusc · 10-03-2009, 06:20 PM

Hi...I make DNA libraries using PE protocol. I modified it a little bit.i do 4 PCR reactions, pool the PCR product and run down the gel again to get rid of any dimers formed.I've started to see a hump fused with my library peak on bioanalyzer, unable to figure out what's causing these humps. I have attached the bioanalyer traces showing a good and a bad library.Please let me know if anybody has seen this problem before and have figured out what's causing these bumps.

Thanks

Susanne · 10-12-2009, 06:07 AM

Hi mollusc, I would assume that the second (right) pic shows an overloaded well. Does it get better if you dilute the sample (1:20 of example)? If you pool the PCR-products and purify then, the conc. should be higher, right - or did you already dilute?

Besides, the hump you see behind the upper marker in both traces is likely ssDNA - biut I guess that one didn't bother you :-)

mollusc · 10-12-2009, 12:41 PM

Hi Susanne,
Thanks for your reply.initially even i thought overloading could be the reason,but then i perfored series of experiments diluting the library and it did give humps again.i just don't have any idea how ssDNA would run on bioanalyzer

Susanne · 10-13-2009, 12:47 AM

Hi, in order not to confuse anything: you are referring to the peak that is larger than 500 bp, the one right behind the large library peak that is disturbing to you, right? And this one didn't go away when diluting the sample?

mollusc · 10-13-2009, 01:16 PM

No, sorry.I'm talking talking about 300bp aterial which i fused with my 400bp library

ArciMol · 05-26-2014, 10:31 AM

mollusc, I have the same problem! There's a hump (median size) fused with my ~400bp library gDNA. I only have this issue with gDNA libraries, not with RNA. I use TruSeq RNA Sample prep kit with both (gDNA sonicated by Covaris previously). It doesn't seem like adapter dimers, since they appear at ~150bp. I really can't figure out what's this strange peak/hump that doesn't align with the reference genome at all. Please, if someone have any ideas, I'd be (very) grateful

Susanne · 06-10-2014, 12:38 AM

Could you share an electropherogram image of the issue, please?

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10-13-2009, 12:47 AM	#4
Susanne Member Location: Germany Join Date: Aug 2009 Posts: 33	Re Hi, in order not to confuse anything: you are referring to the peak that is larger than 500 bp, the one right behind the large library peak that is disturbing to you, right? And this one didn't go away when diluting the sample?

05-26-2014, 10:31 AM	#6
ArciMol Junior Member Location: Chile Join Date: Apr 2014 Posts: 8	mollusc, I have the same problem! There's a hump (median size) fused with my ~400bp library gDNA. I only have this issue with gDNA libraries, not with RNA. I use TruSeq RNA Sample prep kit with both (gDNA sonicated by Covaris previously). It doesn't seem like adapter dimers, since they appear at ~150bp. I really can't figure out what's this strange peak/hump that doesn't align with the reference genome at all. Please, if someone have any ideas, I'd be (very) grateful __________________ Science is ok, but I'm hungry.

10-12-2009, 06:07 AM	#2
Susanne Member Location: Germany Join Date: Aug 2009 Posts: 33	Hi mollusc, I would assume that the second (right) pic shows an overloaded well. Does it get better if you dilute the sample (1:20 of example)? If you pool the PCR-products and purify then, the conc. should be higher, right - or did you already dilute? Besides, the hump you see behind the upper marker in both traces is likely ssDNA - biut I guess that one didn't bother you :-)

10-12-2009, 12:41 PM	#3
mollusc Junior Member Location: cambridge Join Date: Oct 2009 Posts: 9	Hi Susanne, Thanks for your reply.initially even i thought overloading could be the reason,but then i perfored series of experiments diluting the library and it did give humps again.i just don't have any idea how ssDNA would run on bioanalyzer

10-13-2009, 01:16 PM	#5
mollusc Junior Member Location: cambridge Join Date: Oct 2009 Posts: 9	No, sorry.I'm talking talking about 300bp aterial which i fused with my 400bp library

06-10-2014, 12:38 AM	#7
Susanne Member Location: Germany Join Date: Aug 2009 Posts: 33	Could you share an electropherogram image of the issue, please?