QuickExtract DNA extraction: No need to vortex?

[Jing Nie]

Hi All,

When reading Zhang lab CRISPR papers, including the Nature paper they just published today, I noticed that their protocol for QuickExtract genomic DNA extraction protocol is:

65C 15min

68C 15min

98C 10min.

But the protocol from the manufacturer is: (http://www.epibio.com/docs/default-source/protocols/buccalamp-dna-extraction-kit-quickextract-dna-extraction-solution-1-0-catch-all-sample-collection-swabs.pdf?sfvrsn=6)

vortex 15s

65C 6min

vortex 15s

98C 2min.

Did anyone tried extracting the DNA without vortexing? I was just wondering whether the vortexing step is so trivial that they have omitted in their papers?

Thank you!

Best,

Jing

[Le Cong]

Hi Jing,

This particular Quick Extract protocol has indeed been used since our earlier work. We initially were using the standard protocol but then I found this current extraction method (essentially longer time incubation in the thermocycler with no vortexing) can produce PCR-ready genomic DNA of similar quality for routine assay like Surveyor or NGS. Compared with the manufacturer's protocol, which requires pause in the middle to vortex, the one we used will have less hands-on time and no need to pause, so for convenience we've moved to this workflow.

But please bear in mind for cells/tissue that have not been tested in published work, you might need to try it before knowing for sure this will work. And we are happy to discuss if you have specific question for your own experiments.

Hope this helps.

Best,

Le

Le CONG, Ph.D. 丛乐

Broad Institute of MIT and Harvard,

415 Main Street, Cambridge, MA 02142
HHMI International Research Fellow

Tel: (+1) 617-714-7879

Mobile: (+1) 617-823-6132
E-Mail: congl...@gmail.com / co...@broadinstitute.org / con...@mit.edu

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[Jing Nie]

Hi Le,

Thank you so much for your reply! I'll use your protocol for DNA extraction next time. I suspect the manufacturer's vortexing protocol might lead to the shearing of genomic DNA, which might increase the background during Surveyor. 

Congrats on your new Nature paper! 

And hope the CRISPR workshop will go well! I really wished my PI would allow me to go the the workshop, but we are having some budge problems recently... 

Thanks again!

Best,

Jing

On Wednesday, April 1, 2015 at 5:40:06 PM UTC-4, Le Cong wrote:

Hi Jing,

This particular Quick Extract protocol has indeed been used since our earlier work. We initially were using the standard protocol but then I found this current extraction method (essentially longer time incubation in the thermocycler with no vortexing) can produce PCR-ready genomic DNA of similar quality for routine assay like Surveyor or NGS. Compared with the manufacturer's protocol, which requires pause in the middle to vortex, the one we used will have less hands-on time and no need to pause, so for convenience we've moved to this workflow.

But please bear in mind for cells/tissue that have not been tested in published work, you might need to try it before knowing for sure this will work. And we are happy to discuss if you have specific question for your own experiments.

Hope this helps.

Best,

Le

Le CONG, Ph.D. 丛乐

Broad Institute of MIT and Harvard,

415 Main Street, Cambridge, MA 02142
HHMI International Research Fellow

Tel: (+1) 617-714-7879

Mobile: (+1) 617-823-6132
E-Mail: congl...@gmail.com / co...@broadinstitute.org / congl...@mit.edu

[Jean]

On a related note, I am hoping to store my QE extracted DNA for a few days. The Epicentre protocol seems to indicate that we can just freeze the material after Quick Extraction, have you had experience with this? I would be more comfortable if I could purify the DNA out of the QE solution but I can't seem to find a protocol with this. 

Thoughts?

Thank you!

[Le Cong]

Hi Jean,

In our experience it has been safe to store the QE-DNA solution in -20C for extended period of time (>1 year) without losing the ability to use it for PCR-based analysis (Surveyor/NGS). And if just for a few days 4C storage should be sufficient.

Best,

Le

Le CONG, Ph.D. 丛乐

Broad Institute of MIT and Harvard,

Massachusetts Institute of Technology,

415 Main Street, Cambridge, MA 02142

Tel: (+1) 617-714-7879

Mobile: (+1) 617-823-6132
E-Mail: congl...@gmail.com / co...@broadinstitute.org / con...@mit.edu

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