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dA extension of PCR product for TA cloning

dA extension of PCR product for TA cloning - Protocols and Methods Forum

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  #1  
Old 04-02-2009, 10:30 AM
yoginee budhkar
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Default dA extension of PCR product for TA cloning



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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  #2  
Old 04-02-2009, 04:42 PM
Peter Ellis
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Default dA extension of PCR product for TA cloning

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:

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  #3  
Old 04-02-2009, 08:49 PM
WS
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Default dA extension of PCR product for TA cloning

On Apr 2, 12:30*pm, yoginee budhkar <[Only registered users see links. ]> wrote:
Hi,

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

Wo


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