PTALEN_V2 vector

[binnu gangadharan]

Hi

Has anyone used Sanjana et.al 2011 protocol to construct talens? If yes, how do you screen for positive transfections if you have pTALEN_v2(HD/NN/NG/NI) vector backbone? the reason why I am asking is because this vector doesnt have any antibiotic res or eGFP or any other markers to test the efficiency or positive clones in mammalian cell culture(HEK293T).
Please help.

Thanks

Binnu

[Neville Sanjana]

Hi Binnu: These vectors have a hygromycin resistance gene driven by the SV40 promoter. If you prefer to use a different antibiotic selection or a fluorescent marker, you can co-transfect with the appropriate plasmid. I have done this using a separate CMV-EGFP plasmid and it worked great for selection using FACS.

Hope that helps,

- Neville

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[binnu gangadharan]

Dear Neville

Thats great! Do you know what concentration of hygromycin you used for your experiments? Was it done in HEKs?

Thanks
Binnu

[Neville Sanjana]

I co-transfected with a EGFP vector and used FACS, so I have not personally taken advantage of the hygro resistance cassette in that vector. Here's what you can try: Find the lowest possible dose of hygromycin (do a dilution series) that results in no surviving cells after 3-4 days (hygro is a bit slower than puro or blast, so you need to wait this long). Then, after transfection, you can use these conditions to select for a few days. Be careful about leaving on the hygro too long after transient transfection, as you don't want the plasmid to integrate (randomly) into the genome.

Good luck!

- Neville

[binnu gangadharan]

Dear Nivelle

That helps! Will try both methods.

Thanks a lot for your help

-Binnu

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BINNU GANGADHARAN

RESEARCH SCHOLAR

inSTEM, NATIONAL CENTRE FOR BIOLOGICAL SCIENCES

BANGALORE

[binnu gangadharan]

Dear Nivelle

few questions:

which bacterial resistance do pTALEN_v2 vectors have? In the addgene site,they have mentioned it has both Amp and CAT/CmR. I tried doing transformation with both antibiotics together, but couldnt get a single colony. Is it that they have either of these resistance markers?

Also, do you use HEK293 or HEK293T cells for transfection? Could you also brief about the DNA concentrations used in the process.

Sorry for the trouble

Thanks

Binnu

BINNU GANGADHARAN

RESEARCH SCHOLAR

BANGALORE, INDIA
560065