SEQanswers

Go Back   SEQanswers > Sequencing Technologies/Companies > Illumina/Solexa



Similar Threads
Thread Thread Starter Forum Replies Last Post
double peak in mRNA truseq kit ssing Sample Prep / Library Generation 17 03-16-2019 02:00 AM
RNA-seq libraries - double peak after size selection MLog Sample Prep / Library Generation 23 03-08-2012 11:22 AM
Ladder-like Bioanalyzer trace on TruSeq RNA libraries egunth Sample Prep / Library Generation 2 12-01-2011 12:53 PM
truseq libraries for nimblegen catpure mwt General 1 11-18-2011 09:08 AM
Alternative sizing method for 454 PE libraries? CC_seqanswers Sample Prep / Library Generation 12 08-01-2011 07:10 AM

Reply
 
Thread Tools
Old 04-20-2011, 02:28 PM   #1
jlove
Member
 
Location: Boston, MA

Join Date: Apr 2011
Posts: 49
Default TruSeq libraries - double sizing?

Has anyone else been getting PCR products that are exactly double the size of the template? I am seeing this occur with the TruSeq kits and also the KAPA enzyme. I have heard some theories - too much input (seems unlikely), samples clumping together, etc.

I'd love to hear if other people have figured out what is causing this problem. It seems to be a new one over here.

Thanks!
jlove is offline   Reply With Quote
Old 04-21-2011, 05:41 AM   #2
pbluescript
Senior Member
 
Location: Boston

Join Date: Nov 2009
Posts: 224
Default

It is due to overcycling your PCRs, so it could also be due to too much input. I have attached an example of two libraries where I tried different numbers of PCR cycles. The gel shows 6-18 cycles in 2-cycle steps from left to right. In this case, I took the 8 cycle libraries.
Here's another thread talking about this issue:
http://seqanswers.com/forums/showthread.php?t=8894
Attached Images
File Type: jpg PCRovercycling.jpg (7.5 KB, 166 views)
pbluescript is offline   Reply With Quote
Old 04-22-2011, 01:34 PM   #3
jlove
Member
 
Location: Boston, MA

Join Date: Apr 2011
Posts: 49
Default

Update: it was indeed over-cycling that created the problem. I believe that I was getting the amplicons partially hybridizing at the adapter regions and forming Ys which ran larger on the bioA. 10 cycles (vs 18) completely solved the problem. Thanks pbluescript for the ideas!
jlove 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 12:16 AM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2021, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO