Taq polymerase - (Mar/12/2013 )
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?
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.
Normaly, I use
Water 32,6ul
10X Taq buffer 5ul
2mM dNTP mix 5ul
Primer1 1ul
Primer2 1ul
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.
I think it is better to not dilute, anyway if I have to I will do it with buffer 1X
Hope to be usefut
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)
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?
David C H on Tue Mar 12 17:16:00 2013 said:
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?
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
25mM MgCL2 3 ul
Template DNA 2ul