SEQanswers

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



Similar Threads
Thread Thread Starter Forum Replies Last Post
what specific sequence is used for annealing to flow cell? ein_io Illumina/Solexa 3 12-13-2011 07:56 AM
sequencing primer annealing Isabel_Bodmer 454 Pyrosequencing 2 11-18-2011 12:20 PM
Annealing index and primers for multiplexing?? alisrpp Sample Prep / Library Generation 1 03-09-2011 07:51 PM
Annealing index and primers for multiplexing alisrpp Illumina/Solexa 0 02-22-2011 05:12 PM
adapters avatar10 General 2 12-02-2010 05:04 AM

Reply
 
Thread Tools
Old 09-17-2014, 11:21 AM   #1
IdahoRAD
Junior Member
 
Location: Idaho

Join Date: Jan 2014
Posts: 9
Default Annealing adapters

Hello all--

I work on preparing RAD libraries, and have a question regarding annealing adapters.

Suppose my adapters' top and bottom oligos are each at a stock concentration of 100 uM. If I pool each adapter equi-molarly prior to annealing, will I have 100 uM of annealed adapter or will I have 200 uM of annealed adapter?

Of course, I am simplifying my question, because the thing that is confusing me is the molarity-result of pooling equi-molarly. I have searched for an answer, to no avail! I know this is a basic question, but it has really been stumping me!

Thank you,

Cody
IdahoRAD is offline   Reply With Quote
Old 09-17-2014, 10:41 PM   #2
nucacidhunter
Jafar Jabbari
 
Location: Melbourne

Join Date: Jan 2013
Posts: 1,231
Default

You will need concentrated annealing buffer to dilute your oligos for maximum annealing. In the simplest form if you just mix equal volume of 100 uM oligos you will get 50 uM adapters.
nucacidhunter is offline   Reply With Quote
Old 09-18-2014, 08:04 AM   #3
IdahoRAD
Junior Member
 
Location: Idaho

Join Date: Jan 2014
Posts: 9
Default

Nucacidhunter--

Thank you for your reply. Would you mind explaining your rationale, in the example you gave above? Is it because if we pool equi-molarly, we are doubling the volume and thus the molarity decreases?

Suppose I pooled 100 umol of each component of the adapter, will I thus have 100 umol of annealed adapter?

The person who was in charge of adapter preparations at my institution left rather abruptly, so I am trying to figure this out on my own. Seems like with these simple protocols, we (at UIdaho) take them for granted until we have to do them ourselves!
IdahoRAD is offline   Reply With Quote
Old 09-18-2014, 09:46 AM   #4
nucacidhunter
Jafar Jabbari
 
Location: Melbourne

Join Date: Jan 2013
Posts: 1,231
Default

If X mole of oligo A and B is annealed (the same molar concentration) it will give X mole of adapter. In the process the volume has increased by 2x, so the concentration will decrease by 1/2x.
nucacidhunter is offline   Reply With Quote
Old 09-19-2014, 02:14 PM   #5
kerplunk412
Senior Member
 
Location: Bioo Scientific, Austin, TX, USA

Join Date: Jun 2012
Posts: 119
Default

In case you are looking for second opinions, I completely agree with nucacidhunter.
kerplunk412 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 03:55 PM.


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