dA extension of PCR product for TA cloning

[WS]

On Apr 2, 12:30 pm, yoginee budhkar <eeni... from gmail.com> wrote:
> Dear All,
>
> Is it okay to simply replenish the contents like taq buffer and taq
> polymerase in already prepared and stored PCR product (which is not
> purified) to carry out dA extension so that the product becomes useful for
> TA cloning?
>
> This is what I plan to do:
Hi,

you could leave out the second aliquot of Taq buffer. If paranoid, add
a bit of DTT.

Wo

>
> 43 ul of PCR product (not purified, hence already contains salts n other
> buffer components)
> 5 ul of 10X Taq buffer (fresh)
> 1 ul Taq pol (1U)
> 1 ul of dATP (0.5mM final conc)
> 50 ul (total volume)
>
> incubate at 72degree C for 30 min
>
> Will the already present buffer components and salts in the PCR reaction mix
> hamper taq pol activity?
>
> -- Yg