P.S.: D.K.
Why are you just beating about the bush? I had mentioned that I told optimal vector: insert ratio additionally, as your supporters found nothing new in what I said.
And please dont explain to me all that mumbo jumbo of ligation etc.
If it doesn't help you, just don't follow it. It better to start with something that you know than just making a wild guess.
Secondly, if you are really interested on knowing about Avantage and long Pol, just give a google search.
Now, Reitaliating what you said:
If you used proofreading polymerase, it
would have to be A-tailed first. If Taq and the like, no need to.
These days, enzyme mix come with proof reading enzymes in them. therefore, just after PCR,if you want to continue just gel purify the product or if, to be used later, it should be quickly kept in -20, so that proof reading enzyme in that will polish away 3'A left by Taq.
I hope I made myself clear now.
Shifali
On Sun, 28 Dec 2008 DK wrote :
>In article <mailman.1235.1230408039.29717.methods from net.bio.net>, shifali chatrath <shifalich from rediffmail.com> wrote:
> >FYI: Please read, why after PCR, using Taq Pol, Long Pol, Advantage Pol
> >etc. one needs to keep the PCR reaction at -20 quickly?
>>I don't know what Long Pol and Advantage Pol are but as far as
>Taq goes, the answer is clear: no good reason at all!
>> >Also, regarding ligation, refer to Promega's Rapid Ligation buffer literature,
> >they have tested variuos Insert: vector ratio, depending upon insert size,
> >they have got different recombination efficiencies. This ratio was what
> >I told additionally.
>>Just FYI: there is no such thing as optimal vector:insert ratio that
>is applicable in all cases. It all depends on the particular case
>(what is being cloned and how) and ultimate goal (e.g., vector
>self-ligation background important or not, absolute number of
>positive clones important or not, etc).
>>Here is a simple question, see if you can figure it out:
>Ligation 1: Vector only, no insert = 300 colonies (obviously,
>all negative).
>Ligation 2: Vector:insert = 1:4 molar ratio = 400 colonies
>(1 in 6 positive)
>Ligation 3: Vector:insert = 1:50 molar ratio = 70 colonies
>(6 in 6 positive).
>>Can you explain what's going on? And what would you say
>is an optimal vector:insert ratio?
>>DK
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Shifali Chatrath
Graduate Student
Protein science Lab
Dept. of Biological sciences
National University of Singapore
Singapore
+65-65161210
+65-96393449
