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Gewtting the concentration of PCR product and cloning

Gewtting the concentration of PCR product and cloning - Protocols and Methods Forum

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Old 12-26-2008, 02:38 PM
shifali chatrath
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Default Gewtting the concentration of PCR product and cloning



*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 havethat info.

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



On Thu, 25 Dec 2008 Shri wrote :


Shifali Chatrath
Graduate Student
Protein science Lab
Dept. of Biological sciences
National University of Singapore
Singapore
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Old 12-26-2008, 10:17 PM
Aawara Chowdhury
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Default Gewtting the concentration of PCR product and cloning

In <[Only registered users see links. ].net >,
shifali chatrath <[Only registered users see links. ]> wrote:


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).


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 ....".


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