lentiCRISPRv2 protocol (specifically T4 PNK)

[JH]

Hello,

I noticed in the v2 protocol that the T4 PNK has been removed.  Is there a reason for that?

My labmate was following that protocol and couldn't get any transformants, but I tried previously following the v1 protocol (with the T4 PNK) and it worked.

The same oligo annealing/phosphorylation concept should apply, right?

Unless I'm missing something...

[Neville Sanjana]

Hi JH,

The new protocol no longer dephosphorylates the vector and thus does not require phosphorylation of the oligos. The old protocol (which dephosphorylates the vector with AP and then uses T4 PNK to phosphorylate the oligos) will also work just fine with lentiCRISPRv2 and lentiGuide-Puro.

- Neville

--
You received this message because you are subscribed to the Google Groups "Genome Engineering using CRISPR/Cas Systems" group.
To unsubscribe from this group and stop receiving emails from it, send an email to crispr+un...@googlegroups.com.
For more options, visit https://groups.google.com/d/optout.

[Jocelynn Pearl]

I was having some issues with cloning my guide rna into lenticrispr v1 and went ahead and ordered lenticrispr v2.  There were some aspects to the new protocol that I was a bit surprised by- for instance, in a response to another google group conversation about troubleshooting with v1, I was lead to believe that it was absolutely necessary for an efficient cloning to perform the AP and PNK reactions on vector/insert... is it pretty conclusive that these steps are not required with v1 or v2?  Why is there still a 37 degree/ 30 minute incubation step for annealing the oligos if no phosphorylation is taking place?  Would you recommend if I am getting an inefficient cloning ( 2-6 colonies per plate, 0-1 on vector only) to try adding these steps back in?

[Neville Sanjana]

Hi Jocelynn & CRISPR forum friends,

I think you might be using an outdated version of the protocol. (We tried to save folks some $$$ with a non-AP and non-PNK protocol but it doesn't seem to work quite as reliably as the original protocol.) 

Please use this protocol:  http://www.addgene.org/static/cms/files/lentiCRISPRv2_and_lentiGuide_oligo_cloning_protocol_1.pdf

It should result in a very high number of colonies on your positive plate with very little background. It uses AP and PNK, as in the original protocol. 

I think there are a few spots on Addgene's website that still reference the pre-release protocol and we will work with them to update those to the new protocol. Please let me know if you have any questions or difficulty with this sgRNA cloning protocol.

best,

- Neville

[Eric Perkins]

Hi all,

This is Eric, from Addgene. Neville shared the updated protocol with us a few weeks ago, and though we changed it in on some parts of our site, I missed updating the protocol on some of the individual plasmid pages. I apologize for the confusion we caused by that omission. If you have any concerns about data or files on our site, please contact he...@addgene.org.

Regards,
Eric Perkins
Addgene Senior Scientist

[James Cotterell]

hi all i have a quick question also. when i use the protocol without AP/PNK i get no colonies. I switched to using AP and PNk and now i get a couple of colonies. However i'd expect more. Pretty much the only thing i'm doing differently is using the qiaexII gel extraction kit instead of the qiaquick kit. Do you think i should try changing kits for future ligations? i used the qiaexII as it seemed a bit strange to be trying to gel extract a vector >10kb with the qiaquick kit that is supposed to have a very poor recovery rate for that sized DNA fragment.

thanks if you can help

[Neville Sanjana]

Hi James,

QiaexII should work fine for gel extraction. As long as you are able to recover high concentration (via nanodrop or quantitative gel) of digested vector, you should be set.

We typically get way too many colonies so there is definitely something strange going on with your oligo ligation. Have you tested the competency of your cells? Transformation of ligation products is much less efficient than purified plasmid re-transformation, so it is essential to have very competent cells.

Hope that helps,

- Neville

--