Which kits are good for isolating genomic DNA from fixed cells sorted out of a GeCKO screen?

[Mi Cai]

I am planning to do a GeCKO screen with FACS sort. As it takes a long time for sorting through the cells, I would like to fix the cells prior to sorting. However, I have tested our kit for isolating the genomic DNA and PCR sgRNA from both live and fixed cells. The fixed cells gave much lower yield and even the same amount gDNA did not give any PCR product! I am just wondering whether anyone can recommend a kit for isolating gDNA from fixed cells... I used 2% PFA for fixation. Should I lower the PFA concentration?

 

Thanks!

[Neville Sanjana]

Hi Mi,

I've tried several kits from the major manufacturers and didn't like most of them. We have a homemade protocol that is very similar to the modified salting out preparation of genomic DNA. It works great and is MUCH cheaper than any kit. You can find it in the Supplementary Methods to our in vivo GeCKO paper (Chen*, Sanjana* et al., Cell, 2015).

I've attached the detailed protocol here. Hope it helps and let me know if you have any questions.

Neville

On Thu, Apr 7, 2016 at 10:49 AM, Mi Cai <kuaile...@gmail.com> wrote:

I am planning to do a GeCKO screen with FACS sort. As it takes a long time for sorting through the cells, I would like to fix the cells prior to sorting. However, I have tested our kit for isolating the genomic DNA and PCR sgRNA from both live and fixed cells. The fixed cells gave much lower yield and even the same amount gDNA did not give any PCR product! I am just wondering whether anyone can recommend a kit for isolating gDNA from fixed cells... I used 2% PFA for fixation. Should I lower the PFA concentration?

 

Thanks!

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[Ben Boward]

Hi Neville,

Is this method verified for use with fixed cells (pfa)? I wasn't sure because in your manuscript it doesn't seem that you isolated gDNA from fixed cells for gRNA PCR. I am hoping to isolate gDNA and then be able to PCR the gRNA sequences.

Thanks!

Ben

[Ben Boward]

Hi Mi,

Did you ever figure out the best way to isolate gDNA from fixed cells? I tried Neville's method with pretty good results, but I was wondering if you had success with any other methods?

Thanks,
Ben

[Rike]

Hey Mi,

I have tried and compared several kits, even one especially made for fixed cells. They were ok, but not really convincing. So after all the tests I ended up using Trizol, which is the most reliable method for me so far.
Good luck!
Friderike

[Haiyin Liu]

Hi Neville,

Thank you for all your contributions in this forum!

I am hoping to use your homemade method to isolate DNA from our transfected GeCKOv2 library. After making up the solutions, our RNAase solution precipitated heavily when stored at 4ºC (presumably because of the SDS in NK lysis buffer?). Is this acceptable, and is RNAse is still active at 1% SDS?

Best regards,

Haiyin

[Neville Sanjana]

Dear Haiyin,

If you’re referring to the 10mg/ml RNAse solution, yes it does sometimes precipitate. This is fine and it will still be active. I would just do your best to resuspend it before adding to each sample. RNAse is very heat stable (up to 100C!), so you can heat it up a bit to help you resuspend also.

Best,

Neville

[a.ospin...@gmail.com]

Hi Neville,

I just wanted to confirm if this homemade method (from the Cell 2015 paper) is still (up to date) your best recommendation regarding using fixed cells from the screening (you might have found a better way since then). Is 2% PFA the best fixing method or can you recommend something better in this context?

Also, can you comment/predict at all, how well this gDNA extraction method would be compatible with the latest Nature2017 protocol (amplification using NEB next 2x PCR mix, etc)? Of course we are planning to test this, but whatever you can share with us might save days of optimizing.

Thanks heaps,

Alberto

[Neville Sanjana]

Dear Alberto,

Yes, the homebrew gDNA extraction is AWESOME. It outperforms any kit I’ve used and it’s very cheap. I was at a conference yesterday and bumped into someone else using it in San Diego, who also noticed it yields much more DNA than column-based methods. I think it should work fine with lightly PFA fixed tissue.

You can of course use a kit if you prefer (and it will work fine) but I personally use the homebrew gDNA from the Cell 2015 supplementary methods.

Best,

Neville

[Anuja Sathe]

Dear Neville,

Your homebrew method was definitely awesome compared to kits. Thank you for that!

Best,

Anuja.

[wdwl...@gmail.com]

Dear Mi Cai

how you finally extract gDNA from fixed cells, could you please recommend a kit or some methods ?
 
Thanks!

dawei