SEQanswers

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



Similar Threads
Thread Thread Starter Forum Replies Last Post
What is the longest fragment for Illumina Mate pair end seq library? ychang Sample Prep / Library Generation 4 03-08-2012 01:11 AM
Mate-Pair Illumina library problems dottomarco Illumina/Solexa 0 07-27-2011 03:15 AM
Extreme nucleotide bias at fragment ends of Illumina mate pair library kmcarr Sample Prep / Library Generation 3 03-17-2011 02:03 PM
Mate-pair library prep for Illumina bkingham Sample Prep / Library Generation 0 08-09-2010 07:56 AM
Fragment Library vs Mate-pair library magicsiew SOLiD 1 02-03-2010 07:50 PM

Reply
 
Thread Tools
Old 03-01-2011, 07:11 AM   #1
Autotroph
Member
 
Location: Europe

Join Date: Oct 2010
Posts: 22
Default 6kb Mate pair Illumina library

Hi,

Before sending in a sample for 6kb Mate pair library preparation what should be the size range of the DNA?

I have extracted Eukaryotic DNA and run it beside a high range ladder (the highest marker is at 48KB). However,it shows a 'light' smear upto the 20kb marker.

Gel picture attached

Is this DNA too fragmented for Mate pair library prep? I have tried Qiagen kit and phenol chloroform extractions. What is the best protocol to avoid fragmentation?

Thanks
Attached Images
File Type: jpeg gDNA.jpeg (11.0 KB, 88 views)
Autotroph is offline   Reply With Quote
Old 04-19-2011, 07:52 AM   #2
cliffbeall
Senior Member
 
Location: Ohio

Join Date: Jan 2010
Posts: 144
Default

Sorry for the late comment, here's my take.

Phenol extraction is preferable to Qiagen for getting maximum size DNA.

Do not vortex the DNA and use wide bore pipet tips.

That being said, your DNA should be fine as long as the median length is over 6kb.
cliffbeall is offline   Reply With Quote
Old 04-19-2011, 08:39 AM   #3
Autotroph
Member
 
Location: Europe

Join Date: Oct 2010
Posts: 22
Default

thanks...

i did manage to get less fragmented dna with phenol extraction with the pipette tip cut off..
Autotroph is offline   Reply With Quote
Old 04-29-2011, 08:48 AM   #4
pmiguel
Senior Member
 
Location: Purdue University, West Lafayette, Indiana

Join Date: Aug 2008
Posts: 2,317
Default

The pippette tip bore will not make any difference unless you are attempting to isolate DNA >100 kb. The degradation you see is from nucleases present in the cells of of the tissue you are extracting.

Both phenol and qiagen based methods will attempt to rapidly denature/inhibit these nucleases upon cell disruption. Then both methods attempt to fractionate away genomic DNA from the endogenous nucleases (and any other endogenous nucleases.)

I don't thing one method is preferable above the other -- they both have their own intrinsic strengths and weaknesses. But both rely on the sample tissue being intact prior to extraction and on conditions being maintained during sample extraction that prevent nucleases from degrading the genomic DNA.

--
Phillip
pmiguel is offline   Reply With Quote
Old 08-22-2014, 06:05 PM   #5
ArciMol
Junior Member
 
Location: Chile

Join Date: Apr 2014
Posts: 8
Default

Hi everyone! I came a little bit late in the stuff, but... I'm wondering about the high range dna ladder for Mate Pair size selection (I'll use High Range of Thermo Scientific), what voltage (V/cm) and time is enough to separate, at least, the last two bands (~10kb and ~12kb)? I've tried running with the instructions detailed in protocol (3V/cm, for 1,5h) in a 0.4% agarose, but it didn't work, they won't separate well. At last, it gets better with 2,5V/cm, in a 0.3% agarose gel, and running it for... 5,5h!! It's crazyness...
Is there a way to separate the bands as they are in the picture of the protocol? I mean, for real

Thank you in advance!!
__________________
Science is ok, but I'm hungry.
ArciMol is offline   Reply With Quote
Old 08-23-2014, 02:36 AM   #6
nucacidhunter
Jafar Jabbari
 
Location: Melbourne

Join Date: Jan 2013
Posts: 1,238
Default

Quote:
I've tried running with the instructions detailed in protocol (3V/cm, for 1,5h) in a 0.4% agarose, but it didn't work, they won't separate well.
It is not clear what kit you have used and also posting your gel photos would help troubleshooting.
nucacidhunter is offline   Reply With Quote
Old 08-25-2014, 11:35 AM   #7
pmiguel
Senior Member
 
Location: Purdue University, West Lafayette, Indiana

Join Date: Aug 2008
Posts: 2,317
Default

Quote:
Originally Posted by ArciMol View Post
Hi everyone! I came a little bit late in the stuff, but... I'm wondering about the high range dna ladder for Mate Pair size selection (I'll use High Range of Thermo Scientific), what voltage (V/cm) and time is enough to separate, at least, the last two bands (~10kb and ~12kb)? I've tried running with the instructions detailed in protocol (3V/cm, for 1,5h) in a 0.4% agarose, but it didn't work, they won't separate well. At last, it gets better with 2,5V/cm, in a 0.3% agarose gel, and running it for... 5,5h!! It's crazyness...
Is there a way to separate the bands as they are in the picture of the protocol? I mean, for real

Thank you in advance!!
The slower you run it, the better it will work. You can separate 10kb from 8kb on a 0.6% agarose gel by running it very slow -- like for 16 hours (just run it overnight). You want it to be less than 1V/cm. Also, works better if you don't add EtBr to the gel. Stain afterwards instead.

--
Phillip
pmiguel is offline   Reply With Quote
Old 08-26-2014, 10:00 AM   #8
ArciMol
Junior Member
 
Location: Chile

Join Date: Apr 2014
Posts: 8
Default

Thanks Phillip! I was hoping someone would know a faster way to do it, but I knew it was almost impossible for such high sizes. I'm aiming to 12kb - 9kb sizes... so maybe it'll run overnight at 1V/cm.
__________________
Science is ok, but I'm hungry.
ArciMol 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 10:03 PM.


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