Fwd: Gewtting the concentration of PCR product and cloning

[Pow Joshi]

---------- Forwarded message ----------
From: Pow Joshi <pow.joshi from gmail.com>
Date: 2008/12/28
Subject: Re: Gewtting the concentration of PCR product and cloning
To: aawara from pontiff-playground.org



>> > 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.
>>
>> The problem is with your comprehension of English.  Let me clarify:
>>
>> If one wrote, "PCR products .... ARE A-tailed", then it would indicate
>> that products made with a proof-reading polymerase are already A-tailed.
>>
>> On the other hand, if one writes "PCR products .... MUST BE A-tailed",
>> then
>> it indicates that the products made with a proof-reading polymerase are
>> NOT A-tailed, and therefore MUST BE A-tailed for them to be cloned into a
>> TA-vector.
>>
>> Hope that helps you .....
>
>
> I agree with AC .... which is why you need to re-read Dima's reply. You can
> 3' A-tail post-PCR,  if that's what you wish to know., by just incubating
> with regular Taq at 72degC... You would find that in the TOPO manual if you
> read it carefully. It is exactly what DIma said when he wrote " if you used
> proofreading polymerase, it would have to be A-tailed first. If Taq and the
> like, no need to."!!! .....
>
> Sure this is an open forum, and defintely not a place for bashing up, which
> I would say, you were an instigator, a provocateur, with your absolutely
> wrong statement of "What DK says is incorrect!" before you even bothered to
> comprehend what was written (by Dima). We do assume in this forum that
> people understand english as a language. Clearly, though you don't!
>
> Check your premises before you make any statements, is the first rule of
> Logic. You want to read Aristotle perhaps ;)
>
>>
>>
>> AC
>> --
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>
>