SEQanswers

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



Similar Threads
Thread Thread Starter Forum Replies Last Post
Why sub-saturation qPCR in ATAC-seq but not other seq methods? kropak1807 Sample Prep / Library Generation 2 06-21-2017 08:26 AM
ATAC-seq question involving customized primers and adapters duke of iredell Sample Prep / Library Generation 0 04-04-2017 03:07 PM
Cycle number? Indexing/enrichment NEBNext lmw.bio Sample Prep / Library Generation 4 10-26-2015 09:19 AM
Rn/cycles: qPCR decided ATAC-seq library amplification zhaolin Sample Prep / Library Generation 6 01-28-2015 02:23 PM
different PCR cycle number for different microbiomes? petralutra 454 Pyrosequencing 1 08-01-2014 07:58 AM

Reply
 
Thread Tools
Old 11-16-2017, 07:34 AM   #1
JP_seq
Junior Member
 
Location: EST

Join Date: Nov 2017
Posts: 1
Default Atac-Seq - Side qPCR question - cycle number

Hello, I am a new user who has tried searching for an answer to a problem I am having with Atac-Seq library prep.

Let me start off by saying I am using the Buenrostro 2013 Nature Protocols paper and using 50,000 FACS purified mouse cells.

The issue I am having is with the qPCR side reaction requiring MANY more cycles than reported, with others suggesting ~10-15 addition cycles, but my samples requiring ~35-40 extra cycles!

I have attached two images:

(1) Amplification plot showing that I need to add ~37 addition cycles to get 1/3 of the peak fluorescence. You will also notice that the first 30 cycles show almost no amplification and then the reaction takes off -- not sure what this means -- potentially very low starting material? I will note that I decided for these samples to run the qPCR using a 2X SYBR Green Master Mix that contains its own Taq (Thermo Power SYBR Green Master Mix, Cat#4368577) that my lab already had as opposed to the SYBR Green I alone, but otherwise followed everything from the Buenrostro manuscript. I did this because someone else at my institute previous did the same and their side qPCR reactions worked fine and only required ~10 extra cycles. I realize now that this may have been a mistake?

(2) Gel of my amplified final library preps after all PCRs showing that I get a decent smear as reported by some, but don't necessarily have the distinct ~150bp cutoff (sorry for ripping the gel). I end up cutting out 100-1,000bp fragment and gel purifying, followed by PCR purification column, QC, and eventually Seq.


I have yet to send these samples to seq as they were my test samples before I run my more precious samples. I guess the main question I have is: If this was your experiment would you bother sending them to Seq or does taking this many cycles ruin the experiment?


I appreciate any advice the community can offer!

Thanks,

-J
Attached Images
File Type: jpg Amplification Plot.jpg (72.4 KB, 8 views)
File Type: jpg gel.jpg (62.4 KB, 12 views)
JP_seq 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 02:22 PM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2017, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO