dA extension of PCR product for TA cloning

David-Paul Minde via methods%40net.bio.net (by davidminde from gmail.com)
Thu Apr 2 13:34:06 EST 2009


No, (unless you are using a low-quality polymerase with bad proofreading;)
best,
David

2009/4/2 yoginee budhkar <eenigoy from gmail.com>

> 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:
>
> 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
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David Minde MSc (TUM)

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