Gewtting the concentration of PCR product and cloning

[shifali chatrath]

Dear all!
Please read carefully what DK wrote before pointing fingers at me!
Mr. Aawara is also repeating the same thing.[PCR products amplified
with a proof-reading polymerase must be A-tailed ]. Please note that, if the enzyme has proof reading activity, it will remove 3'A and TA cloning cannot be done. And, it is Taq's property to put an extra A at 3' which has nothing to do with its proof reading.

Also, If one has to do A260/A280, one will still be using spectrophotometer and quantifying A260,then why to run standards etc.

P.S.: Dear Vasu!
You can quantitate minute amounts using Nanodrop, you will need at least one microlitre to do that. If you dont have access to nanodrop, then probably, you need to follow the SIMPLE AGAROSE GEL AS SUGGESTED BY DK!
HOpe fully it helps to you.

P.S.: DK's supporters!
This is an open scientific forum and I suppose evrybody has right to give their suggestions as easiest as possible and correct if one thinks that what is written is not what it is! still you can correct me if I wrote anything wrong about proofreading. I am sure, what I wrote is correct.
Don't just bash at someone, as you all are trying to do. I did not rewrite what DK wrote. Rather, the time that you all spent on writing to me, better spend on reading carefully opinions of both of us.

FYI:
Please read ,
why after PCR, using Taq Pol, Long Pol, Advantage Pol etc. one needs to keep the PCR reaction at -20 quickly? 
You will know the difference between what I and DK wrote.

Also, regarding ligation, refer to Promega's Rapid Ligation buffer literature (just a paper,available with Promega), they have tested variuos Insert: vector ratio, depending upon insert size , they have got different recombination efficiencies.
This ratio was what I told additionally, and not just repeating DK's point.
Hopefully, I made myself clear! 
Next time, better check  your "VACUOUS STATEMENTS" !

Sorry to others out of all this!
I know no body is that jobless to poke their noses into this matter.

Shifali

On Sat, 27 Dec 2008 Aawara Chowdhury wrote :
>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
>
>--
>Email: echo 36434455860060025978157675027927670979097959886449930P | dc
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Shifali Chatrath
Graduate Student
Protein science Lab
Dept. of Biological sciences
National University of Singapore
Singapore
+65-65161210
+65-96393449