Testing TALEN function in vitro

[ibrahi...@gmail.com]

Hi
Ive designed TALEN pairs using the golden gate method and sequence verified my product.
I am using a mammalian expression vector when transfecting HEK293T cells using calcium phosphate.
I have two plates, one transfected with TALENs and a repair template with RFP while the other tranfected with the TALENs alone.
I am cutting an important ribosomal protein, supposedly the cells should die without the repair template.
I am posting here because i am failing to see death/RFP signal in my cells.
I am wondering if theres a special protocol for testing TALEN function in vitro (special DNA quantities/transfection method/treatments..)

And how do you guys test if your TALENs are cutting the genome?

Thanks

[mcgillian514]

oh and theres a BFP plasmid there too for a transfection positive control.-i see blue cells everywhere. 

[Chunxin Wang]

Keep in mind that even if your TALEN is working, they won't cut the endogenous locus in all transfected cells and most time they only cut one allele. So, you should not expect to see massive cell death. If you do FACS sorting, you might be able to get a few RFP-positive cells. The intensity of your RFP signal depends on the promoter of your target gene. So, it might be too weak for you to see under microscope but should be picked up by FACS. I would suggest you plate out your transfected cells in 96-well plates with proper dilution to get single colonies and screen for positive clones by PCR.  Black

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[banni...@gmail.com]

Hi there

We do a simple DNA cleavage assay in vitro to test if our TALENs work. We add 1ug of each left and right TALEN plasmid DNA, plus 200-250ng total of a DNA template (at least 1kb with TALEN target site offset from the center by at least 200bp - all to a Quick Transcription / Translation mastermix (TnT system, Promega) and incubate the reaction at 30 degrees for 3 hours, then purify the DNA by phenol-chloroform extraction and run on agarose gels. You clearly see cut target DNA if your TALENs are working. If you want i can send you our more detailed protocol. Our plasmids have an Sp6 promoter site so we use the Sp6 TnT mastermix from Promega for the reaction.
Hope this is helpful,
Cheers
Steph

[Matteo Bovolenta]

Dear Steph,

thank you for sharing your DNA cleavage assay. Could you please send me more informations?

Best Regards,

Matteo

[Bisoso C]

Hi Steph,

 Your cleavage assay seems like a really good way to test even efficiency of the created TALENs. Do you mind posting the detailed protocol on here?

 Thanks.

 B

[liuxian...@gmail.com]

I am doing TALEN work on worms and I am also very interested in the in vitro test for TALEN effect. Your assay design seems to be very nice, could you send me a copy of your protocol?
Thank you so much!

在 2012年11月13日星期二UTC-5下午12时17分19秒，banni...@gmail.com写道：

[Preet]

I liked your DNA cleavage assay to test TALENS.  Would you please share this protocol.  Thanks

[banni...@gmail.com]

Hi everyone, I am happy to share our TnT cleavage assay protocol. But i prefer to email it directly rather than post it to the forum. please feel free to email me your requests: stephanie(dot)bannister(at)univie(dot)ac(dot)at and i will forward you a copy.
Cheers
Steph

[Preet]

On Wednesday, October 31, 2012 1:20:48 AM UTC-4, mcgillian514 wrote:

[Preet]

 

I would like to send you an email for in vitro cleavage assay.  would you give  your email address please.

On Thursday, January 24, 2013 10:36:19 AM UTC-5, banni...@gmail.com wrote:

On Monday, January 14, 2013 10:22:33 PM UTC+1, Preet wrote:

> I liked your DNA cleavage assaI y to test TALENS.  Would you please share this protocol.  Thanks

[Preet]

On Thursday, January 24, 2013 8:36:21 PM UTC-5, Preet wrote:

 

I would like to send you an email for in vitro cleavage assay.  would you give  your email address please.

On Thursday, January 24, 2013 10:36:19 AM UTC-5, banni...@gmail.com wrote:

On Monday, January 14, 2013 10:22:33 PM UTC+1, Preet wrote:

> I liked your DNA cleavage assay to test TALENS.  Would you please share this protocol.  Thanks

[zhaoxue...@gmail.com]

Hi, Dear Steph,

I also like your DNA cleavage assay to test TALENS.  Would you please share this protocol.  Thanks 

My e-mail: zhaoxue...@gmail.com.

在 2012年10月31日星期三UTC-4上午1时20分48秒，Ibrahim Kays写道：

[Danioivanio]

Hey, great Idea with the TnT Assay, can you forward me the protocol?? Pls. Thank you very much. Best Ivan

[Danioivanio]

Hey, I am not sure if posting below was correct. Anyway regarding the TnT Assay, can you Pls forward me the protocol, would be great! Thanks Ivan

[wingl...@gmail.com]

Hi Steph

I also liked your in vitro DNA cleavage assay could you email me the protocol.

Thanks for sharing

Wing

[zhangluj...@gmail.com]

Dear Steph,

Thank you for sharing your DNA cleavage assay. It sounds great! I liked your DNA cleavage assay to test TALENS. Would you please email me this protocol? Thanks! My email address: zhangl...@163.com

[朱广晶]

HI, Steph,

Could you please send me a copy of your protocol as well? My email address is johnt...@gmail.com

Thanks a lot.

Guangjing

On Tuesday, November 13, 2012 12:17:19 PM UTC-5, banni...@gmail.com wrote:

[banni...@gmail.com]

Please see my message earlier in this feed for my email address and I will send you the protocol. - It's easier to keep track of who i already sent the protocol via email than on the group feed.

Cheers
STeph

[qiuj...@gmail.com]

Hi Steph,

I'm trying to test my TALENs function.
Could you please share your protocol to me?

Many thanks
JQ

[Filip Weisz]

Hi Steph, 
Could you please send me more informations about DNA cleavage assay? And do you use T7E1 assay? 

Thank you.
Dne úterý, 13. listopadu 2012 18:17:19 UTC+1 banni...@gmail.com napsal(a):

[gurumoorthy.k...@googlemail.com]

Hi,
i also liked your in vitro assay. Could you please send me the protocol?
thanks
GK

[amogh nair]

Apr 5

Dear Steph,

Thank you for sharing your DNA cleavage assay. It sounds very good! I liked your DNA cleavage assay to test TALENS.  Would you please email me this protocol? Thanks! We are also planning to knockdown a gene in human embryonic stem cells as well. I would appreciate if you could help me share a procol for human cells as well..

My email address:amog...@gmail.com.

Regards,

Amogh Nair.

[lina W]

Hi Steph,
I would also be very interested to get a detailed protocol from the DNA cleavage assay you've established.
Thanks a lot for your help!
Greetings,
Lina

[banni...@gmail.com]

Hi there,

I had left my email address on this thread so people could email me directly for a copy of the protocol. I guess the post got taken away. Here it is again for anyone who wants a copy of the protocol. I just want to email it separately rather than posting on the site because it's quite long, and we haven't published yet, so we would like to be able to keep track of whom we share the protocol with, so that when we publish we can forward appropriate citation details to everyone.

The email you can contact me on for the protocol is:

stephanie"dot"bannister"at"univie"dot"at"dot"ac (sorry for the cryptic long-hand but maybe this will stop the message being deleted.

Cheers
Steph

[MARK Smith]

I also like your DNA cleavage assay to test TALENS.  Would you please share this protocol.  Thanks 

My e-mail: minei...@gmail.com

Best

Ibrahim Kays於 2012年10月31日星期三UTC+8下午1時20分48秒寫道：

[torfa...@gmail.com]

Dear Steph,

I am trying to test my TALENS result in HEK293FT following BES transfection protocol. I have designed DNA primers and I am going to cut my PCR product with FokI. Could you please send me a copy of your protocol?

Kind regards,

[stuar...@hotmail.com]

On Wednesday, October 31, 2012 6:20:48 AM UTC+1, Ibrahim Kays wrote:
> Hi
> Ive designed TALEN pairs using the golden gate method and sequence verified my product.
> I am using a mammalian expression vector when transfecting HEK293T cells using calcium phosphate.
> I have two plates, one transfected with TALENs and a repair template with RFP while the other tranfected with the TALENs alone.
> I am cutting an important ribosomal protein, supposedly the cells should die without the repair template.
> I am posting here because i am failing to see death/RFP signal in my cells.
> I am wondering if theres a special protocol for testing TALEN function in vitro (special DNA quantities/transfection method/treatments..)
>
> And how do you guys test if your TALENs are cutting the genome?
>
> Thanks

HI, Could you please send me the protocol, thanks

Stuart

[Mingxiu Wang]

Hi, Dear Steph, 

I also like your DNA cleavage assay in vitro to test TALENS.  Would you please share this protocol with me.  Thank you very much！

   My email address is wangmin...@163.com.

[Mingxiu Wang]

Dear Steph,

I am very interested in your DNA cleavage assay in vitro to test TALENs. It is really a simple method and I am appreciated with your creative ability.

So I was wondering if you could send me your detailed protocal.

Mingxiu Wang

Wuhan Institute of Virology, Chinese Academy of Science

Marth 8th, 2014

在 2012年11月14日星期三UTC+8上午1时17分19秒，banni...@gmail.com写道：

Hi there

We do a simple DNA cleavage assay in vitro to test if our TALENs work. We add 1ug of each left and right TALEN plasmid DNA, plus 200-250ng total of a DNA template (at least 1kb with TALEN target site offset from the center by at least 200bp - all to a Quick Transcription / Translation mastermix (TnT system, Promega) and incubate the reaction at 30 degrees for 3 hours, then purify the DNA by phenol-chloroform extraction and run on agarose gels. You clearly see cut target DNA if your TALENs are working. If you want i can send you our more detailed protocol. Our plasmids have an Sp6 promoter site so we use the Sp6 TnT mastermix from Promega for the reaction.
Hope this is helpful,
Cheers
Steph

[banni...@gmail.com]

On Wednesday, October 31, 2012 6:20:48 AM UTC+1, Ibrahim Kays wrote:
> Hi
> Ive designed TALEN pairs using the golden gate method and sequence verified my product.
> I am using a mammalian expression vector when transfecting HEK293T cells using calcium phosphate.
> I have two plates, one transfected with TALENs and a repair template with RFP while the other tranfected with the TALENs alone.
> I am cutting an important ribosomal protein, supposedly the cells should die without the repair template.
> I am posting here because i am failing to see death/RFP signal in my cells.
> I am wondering if theres a special protocol for testing TALEN function in vitro (special DNA quantities/transfection method/treatments..)
>
> And how do you guys test if your TALENs are cutting the genome?
>
> Thanks

Hi Everyone,

I'm happy to say our paper on establishing TALENs for targeted genome engineering in the marine annelid worm Platynereis dumerilii is now online as an early edition Author Manuscript [Genetics, early online, March 20th 2014]. A warning though, there are some problems with the PDF conversion for the figures in some internet browsers. So far only Google Chrome allows for the figures to be viewed in complete form - the publishers are working on trying to fix this for other browsers though.

You can also download a full-text PDF copy of our paper, plus our full in vitro TnT assay protocol for testing TALEN function in vitro from my ResearchGate page: https://www.researchgate.net/publication/260999236_TALENs_Mediate_Efficient_and_Heritable_Mutation_of_Endogenous_Genes_in_the_Marine_Annelid_Platynereis_dumerilii#share

The protocol is a "publication resource" linked to the paper.

Hope this is helpful to those of you who are interested in testing TALEN functions in vitro.

For others who already got the protocol by email I will send you the full citation details once the paper is out in print in May.

Cheers & Happy TALEN making and testing!
Steph

[francis...@gmail.com]

Hi,

Very nice approach! Is there someone who used a similar approach to test Crispr/Cas activity? Because there you have different promoters (CBh and U6).

Cheers,

Francis

Op vrijdag 4 april 2014 10:52:11 UTC+2 schreef banni...@gmail.com:

[Carla Siniscalchi]

Hi Stephan,
I like your DNA cleavage assay, could you please send me all protocol?
Thanks.

carla.si...@gmail.com

[wangz...@126.com]

Hi Steph,
I am Zhuo, I want to use your protocol to test my TALEN plasmids in vitro, would you to share the protocol with me, Thanks!

zhuo

在 2012年11月14日星期三UTC+8上午1时17分19秒，banni...@gmail.com写道：
> Hi there
>
> We do a simple DNA cleavage assay in vitro to test if our TALENs work. We add 1ug of each left and right TALEN plasmid DNA, plus 200-250ng total of a DNA template (at least 1kb with TALEN target site offset from the center by at least 200bp - all to a Quick Transcription / Translation mastermix (TnT system, Promega) and incubate the reaction at 30 degrees for 3 hours, then purify the DNA by phenol-chloroform extraction and run on agarose gels. You clearly see cut target DNA if your TALENs are working. If you want i can send you our more detailed protocol. Our plasmids have an Sp6 promoter site so we use the Sp6 TnT mastermix from Promega for the reaction.
> Hope this is helpful,
> Cheers
> Steph
>
>
>