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Old 06-22-2012, 06:14 AM   #1
Derek Daly
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Location: Liverpool UK

Join Date: Jun 2012
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Default Nextera DNA sample preperation problem

Hello everyone my first time on here
Can any one help
Im using the Nextera sample preperation kit.
My problem in some samples
the DNA does not sheer properly leaving me with very large Fragments well over 1kb

The DNA is WGA samples from single cells

Has anyone encountered this problem and if so did yoou find a solution


Hope you can help Derek
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Old 06-22-2012, 07:56 AM   #2
Bucky
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Hey Derek,

How do you quantify your amount of input DNA, maybe you are putting in too much DNA.
You can also try to use less input DNA. Try to set up a reaction with 25ng of input DNA.

Hope that helps.
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Old 06-26-2012, 06:29 PM   #3
weigrc
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May be caused by the volume you applied, like using Covaris, some unsuitable volumes in the Covaris shearing step can cause the air gap contributing to "phase separation" that results in the presence of larger fragments in the sheared DNA.

Last edited by weigrc; 06-26-2012 at 07:17 PM.
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Old 06-27-2012, 04:32 AM   #4
pmiguel
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Quote:
Originally Posted by weigrc View Post
May be caused by the volume you applied, like using Covaris, some unsuitable volumes in the Covaris shearing step can cause the air gap contributing to "phase separation" that results in the presence of larger fragments in the sheared DNA.
Nextera uses a "tamed" transposase to break strands and add end-adapters. So air gaps will not be an issue.

The WGA is probably the issue. Most of them create complex branched double-stranded structures of extremely high molecular weight. My wanton speculation would be that the transposase likes the WGS junction points where there is a single-stranded section? Then those areas would get hammered while the double stranded segments would get few insertions.

Seems like you could overcome this bias (or most other sorts of bias) by increasing the incubation time.

--
Phillip
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Old 06-27-2012, 07:58 AM   #5
Derek Daly
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Location: Liverpool UK

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Default Derek

Hi everyone thanks for the replies
I know my DNA conc was correct
We thought here in Liverpool it was a WGA problem as pmiguel points out
Epi center suggest its a phenomenom called bird nesting were the samples are sticking together and look like longer fragments on bioanalyser traces when in fact they are the correct size.

They suggest that the denaturning step during bridge PCR will eliminate the tangle and the average size will settle back to 200 400bp

if you interseted you can find it here

http://epicentral.blogspot.co.uk/201...irds-bird.html

thanks for your help guys

ta Derek
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