pCS2Tal3 target vectors

[KerryGirleen]

Hi all

I'm looking to use pCS2Tal3 as my final target vector for my TALENs
but I've found that there are two mentioned in most protocols and on
the ADDGENE website:

pCS2TAL3DD and pCS2TAL3RR.

Does it matter which one I use? Can I use the same one for my forward
and reverse monomers or do I need to use both? If not which one would
you recommend?

Thanks
Miriam

[Richard Maguire]

Hi, Miriam.

The Grunwald lab pCS2 TAL3 destination vectors use obligate
heterodimeric FOKI domains. DD must heterodimerise with RR in order to
induce a double strand break. This reduces possible off target effects
you may otherwise get due to spurious homodimerisation of the "standard"
homodimeric FokI.

I presume you're using the Golden Gate method, so simply clone your
completed Left (forward) array into DD and the Right (reverse) TAL
array into RR. The order is important, as another feature of the
Grunwald pCS2TAL3 plasmids are an optimised linker length between the
TAL array and FOK1. This is related to the "spacer" length you leave in
between the target arrays and has a large impact on the efficiency at
which the double strand breaks occur.

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--
Dr Richard Maguire
Postdoctoral Research Associate
Pownall Lab
University of York
Department of Biology (AREA 11)
Wentworth Way
Heslington
York
YO10 5DD
United Kingdom

Tel: +44 (0) 1904 328689
Fax: +44 (0) 1904 328505

[KerryGirleen]

On Thursday, 9 May 2013 09:47:48 UTC+1, Richard Maguire wrote:
> Hi, Miriam.
>
>
>
> The Grunwald lab pCS2 TAL3 destination vectors use obligate
>
> heterodimeric FOKI domains. DD must heterodimerise with RR in order to
>
> induce a double strand break. This reduces possible off target effects
>
> you may otherwise get due to spurious homodimerisation of the "standard"
>
> homodimeric FokI.
>
>
>
> I presume you're using the Golden Gate method, so simply clone your
>
> completed Left (forward) array into DD and the Right (reverse) TAL
>
> array into RR. The order is important, as another feature of the
>
> Grunwald pCS2TAL3 plasmids are an optimised linker length between the
>
> TAL array and FOK1. This is related to the "spacer" length you leave in
>
> between the target arrays and has a large impact on the efficiency at
>
> which the double strand breaks occur.
>
>
>
>

Hi Richard

Thanks for clearing that up for me!

Miriam