Gewtting the concentration of PCR product and cloning

Aawara Chowdhury via methods%40net.bio.net (by aawara from pontiff-playground.org)
Fri Dec 26 17:17:21 EST 2008


In <mailman.1233.1230315276.29717.methods from net.bio.net>,
 shifali  chatrath <shifalich from rediffmail.com> wrote:

>  ?Hi!
> I want to correct DK's point. If it is proofreading polymerase, 3'A will get depleted off as a result of proof reading. Taq Pol/ Long Pol amplified products can generally be used for TA cloning. Their fact will have that info.

How are you correcting what Dima posted?  You're simply repeating
what he wrote (which was correct to begin with - PCR products amplified
with a proof-reading polymerase must be A-tailed to be TA-cloned).

> Secondly, to me the suggestion to Point 2 is tedious. 

Why is it tedious?  Dima gave two methods to quantify PCR products - a)
estimate their concentration by comparing band intensity to a marker of
known concentration, or b) obtain a A260/A280 ratio by spectrophotometry.

You've provided no method - other than the vacuous statement "Gel purified
PCR product can be directly quantified ....".

> Gel purified PCR product can be directly quantified and ligation reaction can be set up, depending upon the size of the insert, one needs to clone. 5:1::insert:vector is the optimal molar ratio for cloning insert of 1250bp. Just quantify the insert and go ahead.
> All the best.
>
> Shifali Chatrath
> Graduate Student
> Protein science Lab
> Dept. of Biological sciences
> National University of Singapore
> Singapore
> +65-65161210
> +65-96393449

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