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Taq polymerase


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7 replies to this topic

#1 sercanpazarlar

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Posted 12 March 2013 - 01:44 AM

Hi

I am trying to optimised RT-PCR for my lab. I need to dilute Taq DNA Polymerase.

Standart 5U/ul and I need 3U/ul.

How can I do this? and with water or else?

#2 leelee

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Posted 12 March 2013 - 03:57 AM

Why dilute it? Why not just add a smaller amount to your master mix to get the desired number of units per reaction?

I don't think it is a good idea to dilute a stock of an enzyme.

#3 sercanpazarlar

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Posted 12 March 2013 - 05:39 AM

Normaly, I use
Water 32,6ul
10X Taq buffer 5ul
2mM dNTP mix 5ul
Primer1 1ul
Primer2 1ul
Taq DNA polymerase 0.4 ul
25mM MgCL2 3 ul
Template DNA 2ul

my prof tell me to change the Taq DNA concentrations to 3U/ul, 1U/ul, 0.5U/ul, 0.1U/ul and 0.01 U/ul
my stock is 5U/ul
So its hard to arrange the amounts exactly changing 3U, ...... That was the idea diluting is better.

#4 criscastells

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Posted 12 March 2013 - 08:44 AM

I think it is better to not dilute, anyway if I have to I will do it with buffer 1X

Hope to be usefut

#5 David C H

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Posted 12 March 2013 - 09:16 AM

Enzyme storage buffers are often 50% glycerol with whatever buffer makes the enzyme happy. If you bought your taq, it should come with a manual or product information that lists the storage buffer (or you should be able to find this on their website). For example, Roche includes this with their taq: Enzyme storage buffer: 20 mM Tris-HCl, 1 mM DTT, 0.1 mM EDTA, 0.1 M KCl, 0.5% Nonidet P40 (v/v), 0.5% Tween 20 (v/v), 50% glycerol (v/v), pH 8.0 (4°C)

#6 leelee

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Posted 12 March 2013 - 09:46 PM

I agree with David in that you should use the same buffer if you would like to dilute your enzyme.

But... I don't think messing around with enzyme amounts is the best approach for optimising a PCR. What is your professor's rational for looking at the enzyme amount for optimising?

#7 sercanpazarlar

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Posted 12 March 2013 - 10:26 PM

Enzyme storage buffers are often 50% glycerol with whatever buffer makes the enzyme happy. If you bought your taq, it should come with a manual or product information that lists the storage buffer (or you should be able to find this on their website). For example, Roche includes this with their taq: Enzyme storage buffer: 20 mM Tris-HCl, 1 mM DTT, 0.1 mM EDTA, 0.1 M KCl, 0.5% Nonidet P40 (v/v), 0.5% Tween 20 (v/v), 50% glycerol (v/v), pH 8.0 (4°C)


Thats what I think.

On other opinion without diluting
if I use 0,4ul Taq that means I use 2U ?
or if I use 2ul that means 10U?

#8 sercanpazarlar

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Posted 13 March 2013 - 04:46 AM

Hi again,

The file that was attached is a research I found. It shows exactly what I want to do.
There are Taq DNA polymerase concentrations (Unite uL-1)

I would like to do this but I dont know how.
I have Taq 5U. And use the protocol ;

Water 32,6ul
10X Taq buffer 5ul
2mM dNTP mix 5ul
Primer1 1ul
Primer2 1ul
Taq DNA polymerase 0.4 ul
25mM MgCL2 3 ul
Template DNA 2ul

Attached Thumbnails

  • taq.jpg





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