SEQanswers

Go Back   SEQanswers > Applications Forums > Sample Prep / Library Generation



Similar Threads
Thread Thread Starter Forum Replies Last Post
loss of library DNA in the last purification, need help Anjani Sample Prep / Library Generation 3 06-26-2014 11:52 PM
Library prep with <100ng DNA? sstarkenburg Illumina/Solexa 20 03-05-2013 04:25 AM
Can Truseq DNA library get >1ug DNA library at last? rnal Illumina/Solexa 2 04-12-2012 07:36 AM
how much DNA is required for library preparation? chromatin Sample Prep / Library Generation 0 11-14-2010 06:46 PM
Library prep using chromosome sorted DNA Scotch Sample Prep / Library Generation 0 08-14-2010 04:44 PM

Reply
 
Thread Tools
Old 10-03-2009, 05:20 PM   #1
mollusc
Junior Member
 
Location: cambridge

Join Date: Oct 2009
Posts: 9
Default Shoulders/humps seen in DNA library

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
Attached Images
File Type: jpg Bioanalyzer.jpg (35.0 KB, 175 views)
mollusc is offline   Reply With Quote
Old 10-12-2009, 05:07 AM   #2
Susanne
Member
 
Location: Germany

Join Date: Aug 2009
Posts: 33
Default

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 :-)
Susanne is offline   Reply With Quote
Old 10-12-2009, 11:41 AM   #3
mollusc
Junior Member
 
Location: cambridge

Join Date: Oct 2009
Posts: 9
Default

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
mollusc is offline   Reply With Quote
Old 10-12-2009, 11:47 PM   #4
Susanne
Member
 
Location: Germany

Join Date: Aug 2009
Posts: 33
Default 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?
Susanne is offline   Reply With Quote
Old 10-13-2009, 12:16 PM   #5
mollusc
Junior Member
 
Location: cambridge

Join Date: Oct 2009
Posts: 9
Default

No, sorry.I'm talking talking about 300bp aterial which i fused with my 400bp library
mollusc is offline   Reply With Quote
Old 05-26-2014, 09:31 AM   #6
ArciMol
Junior Member
 
Location: Chile

Join Date: Apr 2014
Posts: 8
Default

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.
ArciMol is offline   Reply With Quote
Old 06-09-2014, 11:38 PM   #7
Susanne
Member
 
Location: Germany

Join Date: Aug 2009
Posts: 33
Default

Could you share an electropherogram image of the issue, please?
Susanne is offline   Reply With Quote
Reply

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off




All times are GMT -8. The time now is 06:05 AM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2020, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO