dA extension of PCR product for TA cloning

yoginee budhkar via methods%40net.bio.net (by eenigoy from gmail.com)
Thu Apr 2 05:30:26 EST 2009


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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