dA extension of PCR product for TA cloning

[Peter Ellis]

Why would you want to add more of the buffer salts?  It's not like 
magnesium, sodium and chloride ions can "go off" over time.  In theory you 
could just add fresh nucleotides (in case the original PCR reaction 
depleted them too severely) and some Taq.

However, ask yourself this question:  why are you doing an A-tailing 
reaction?

Presumably the answer is because the polymerase you used to do the PCR 
either does not add A-tails, or (likely for many polymerase mixes) it has 
3' to 5' exonuclease activity and in fact removes them.

In the latter case, then there's no point adding A-tails without purifying 
it, as any remaining enzyme from the initial PCR will just chew the tails 
off again.

Purify it before you A-tail it - it only adds a few minutes and takes all 
the unknowns out of the equation.

Peter


On Thu, 2 Apr 2009, yoginee budhkar 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:
>
> 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
>