SEQanswers

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



Similar Threads
Thread Thread Starter Forum Replies Last Post
Another question about Bridge amplification/insert size chenbati Illumina/Solexa 13 10-20-2016 08:41 AM
Insert size != Fragment size? Boel Bioinformatics 6 12-12-2013 09:28 AM
insert size question for sequencing xp8 Illumina/Solexa 2 08-25-2011 08:21 AM
About Insert, Insert size and MIRA mates.file aarthi.talla 454 Pyrosequencing 1 08-01-2011 02:37 PM
Large insert libraries busypops Illumina/Solexa 3 05-03-2011 11:07 AM

Reply
 
Thread Tools
Old 06-02-2011, 01:39 PM   #1
ZAAB
Member
 
Location: Boston

Join Date: Apr 2011
Posts: 18
Default 150nt insert size, generating libraries, question!

Hi All:
I'm preparing libraries for the HiSeq...
We're generating a library of variants, and the whole construct is 143nt long...with 30 nt in the middle that are variable (lots of variants!).

Trying to adapt the TruSeq protocols to my application, I've already had to resort to using Qiagen Minelute columns instead of AmpPure (my size is below the threshold (all in the wash)).

I am planning on producing 1ug of DNA (via PCR, unfortunately) to then do end-repair, phosphorylation, ligation, etc...

Am I on the right track here...or should I assume loss after shearing? By 'loss', I mean that not all gDNA is at the correct size after shearing, but I know my PCR'd DNA is All the right size...or do I just go forward with 1ug of PCR'd DNA?

Also, since my final size is going to be smaller than suggested, should I alter amplification/extension conditions in the cluster station? I'm not running the machine, so what should I tell the tech?

Thanks!
ZAAB is offline   Reply With Quote
Old 06-02-2011, 04:24 PM   #2
shurjo
Senior Member
 
Location: Rockville, MD

Join Date: Jan 2009
Posts: 126
Default

Quote:
We're generating a library of variants, and the whole construct is 143nt long...with 30 nt in the middle that are variable (lots of variants!).
If all your contructs are the same size, why do you need to shear? In fact, if you used Taq for the PCR, you can go straight to adapter ligation afterwards.

Just my two cents.
shurjo is offline   Reply With Quote
Old 06-03-2011, 05:03 AM   #3
ZAAB
Member
 
Location: Boston

Join Date: Apr 2011
Posts: 18
Default

Exactly! Yes, I am using Taq, and Yes, I do not need to shear...
Just interested in the amount of DNA from a 1ug shearing 'reaction'..what amount is of the correct size...80%? I'll have 100%, so I don't want to overload the downstream analysis...

Thanks for confirming why I don't need to do endrepair, etc.

Z
ZAAB is offline   Reply With Quote
Old 06-03-2011, 06:51 AM   #4
HESmith
Senior Member
 
Location: Bethesda MD

Join Date: Oct 2009
Posts: 509
Default

All of the input DNA is present through the gel extraction step, so all of the reaction conditions are calculated for that amount. It's immaterial what fraction is of the desired size until the PCR amplification, and at that point the protocol has recommended guidelines based on the amount of fragment.
HESmith is offline   Reply With Quote
Old 06-03-2011, 07:12 AM   #5
ZAAB
Member
 
Location: Boston

Join Date: Apr 2011
Posts: 18
Default

I guess I was worried about overloading the ligation reaction with insert. I am never convinced that all DNA is carried forward when there is a wash/Ampure step.
Thanks for all replies.

I ended up performing the A-tailing rxn again, even though its TAQ-amplified, as I know A's do 'fall off' from experience. And, that volume and enzyme mix is present in the ligation rxn, with no cleanup involved between those steps.

THanks!
ZAAB 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 06:58 PM.


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