6kb Mate pair Illumina library

[Autotroph]

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

[cliffbeall]

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.

[Autotroph]

thanks...

i did manage to get less fragmented dna with phenol extraction with the pipette tip cut off..

[pmiguel]

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

[ArciMol]

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!!

[nucacidhunter]

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.

[pmiguel]

Originally Posted by ArciMol

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

[ArciMol]

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.