lentiCRISPRv2 digestion

[DHC]

I am trying to perform the digestion to the lentiCRISPRv2 plasmid but I am having a lot of problems.  I am doing the same reaction as in the Zhang lab protocol but without AP treatment just to see if the plasmid can cut.  I left the reaction for 30 minutes, 1 hour and I even left it for 9 hours but I still don't have the 2KB band.  Any ideas?

[A Duckworth]

Hi, Which buffer do you use with the restriction enzyme and which restriction enzyme are you using (should be BsmBI)?

Are you following the LENTICRISPR protocol and not the PX330 one? Neither protocol requires you to use AP now. 

You should be following this protocol for LENTICRISPRv2 -> http://www.addgene.org/static/cms/files/lentiCRISPRv2_and_lentiGuide_oligo_cloning_protocol.pdf

Follow this single-step digestion-ligation protocol for PX330, PX458 or PX459 -> http://www.genome-engineering.org/crispr/wp-content/uploads/2014/05/CRISPR-Reagent-Description-Rev20140509.pdf

Hope this helps

Andrew

[Neville Sanjana]

Hi all,

For your digest, please make sure you include DTT with the BsmBI enzyme, as DTT is a necessary co-factor in the reaction. The Addgene protocol listed there is unfortunately an old version that we have had trouble getting Addgene to take down. The correct version is linked on the Addgene page for lentiCRISPRv2 plasmid and on our website:

http://genome-engineering.org/gecko/?page_id=15

- Neville

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[Eric Perkins]

Hi all, this is Eric from Addgene. As Neville mentioned, we had a difficult time scrubbing that old protocol out of our system, and though we haven't linked to it from our site in quite some time, Google and other search engines were still finding it. One of our developers spent a good part of his day replacing that old protocol with the new one. As of now, I think we've purged the system of the older protocol that did not mention DTT in the BsmBI digest components. Please contact me directly if you stumble upon one of the older protocols.

On behalf of Addgene, I would like to apologize for time and productivity lost due to this error. Though the links from our own website were fine, this issue has taught us an important lesson about how Google finds and caches files that wouldn't otherwise be available publicly. I'm optimistic that the problem has been resolved now. Good luck with your CRISPR experiments!

Regards,

Eric

[Neville Sanjana]

Hi Eric,

Thank you for following the posts here and helping us out. We really appreciate Addgene updating that link (as you can see, many folks are using the oligo protocol from that link). 

Looks great to me and I hope everyone's lentiCRISPR cloning goes well with the updated protocol!

Thanks again,

- Neville

[A Duckworth]

thanks for the update.  My experiment is working with the protocol I was using, all be it with only 10 colonies per plate.  Ill update to the latest protocols and see if I can achieve more.

regards

Andrew

[Joydeep Bhadury]

hi
the digestion for me works fine but i too get around 10 colony per plate at the max.i tried using stable3 and xl10 gold but same result in both cases..is there any similar problem faced by others.,in that cases i would like to know if you could troubleshoot the same. .

regards
joydeep

[Neville Sanjana]

Hi Joydeep and Andrew,

You might want to try Zymo's Z Comptentent Cells (now called Mix & Go, I think). It allows you to cheaply make your own Stbl3 and results in ultracompetent cells that perform better than commercial Stbl3 in our hands. We typically see hundreds of colonies in the oligo cloning protocol using these cells (with near 0 on the negative control plate).

Hope that helps,

- Neville