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Old 03-29-2017, 05:54 AM   #1
IanAWarren
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Location: Cambridge

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Default High Molecular Weight genomic DNA extraction

Hello,

Our lab is looking into doing some high molecular weight DNA extractions for use with the latest nanopore sequencing instruments.

Does anyone have any recommendations for the best method?

We are looking at a kit from Qiagen, Agilent and a DNAzol based method, here are the links:

Qiagen:

https://www.qiagen.com/us/shop/sampl...00g/#resources

Agilent:

http://www.genomics.agilent.com/en/N...bId=AG-PR-1168

DNAzol:

http://biorxiv.org/content/early/2015/05/20/019281


Thank you in advance for any help that you can provide!

Ian
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Old 03-29-2017, 07:20 AM   #2
lorendarith
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I think a lot of people are going back to "old school" protocols, which seem to yield better results and longer reads, instead of using kits.

Here some examples:
https://www.protocols.io/groups/mini...-on-their-mind
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Old 03-29-2017, 08:28 AM   #3
ATϟGC
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The best method might depend largely on the taxon or taxa you are using if there are abundant co-precipitating contaminants. I have attached a method I developed/modified for HMW extraction of DNA from polysaccharide-rich tissues for PacBio sequencing (DOI:10.1111/1755-0998.12616)

I have not tried either of the two protocols described below but they might also be of interest to you:

http://enseqlopedia.com/2017/03/hmw-...e-10xgenomics/
Attached Files
File Type: pdf Steeves et al_MER-MoLSC_HMW_DNA_extraction_method.pdf (134.1 KB, 19 views)
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Old 03-30-2017, 07:27 AM   #4
IanAWarren
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Thank you very much for the responses. I will give these protocols a go and see what happens.

Fingers crossed (when not pipetting!)
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Old 04-22-2017, 01:56 PM   #5
Carcharodon
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I've heard of a lot of folks having good success with phenol-chloroform extractions. They report high yields of high-quality, high-weight gDNA.

One thing we like to do with the Qiagen Kits (DNeasy Blood and Tissue) is a triple-elution. We use 35uL water or buffer to elute some DNA, then 50uL water or buffer to elute more DNA, and finally do 2x 50uL elutions.

The first elution (35uL) tends to have most of the "sheared" DNA - those small fragments are washed out.

The second elution (50 uL) tends to have high-weight DNA, but may have some smaller/sheared fragments.

The third elution (100 uL) tends to have nothing but high-weight gDNA, though you may find the yield is lower than in the other two elutions.

If need be, you could combine the second and third elutions to achieve the desired mass of gDNA.

For the second two elutions (elution 2 and elution 3): you may consider warming your water/buffer to 70 degrees C.
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