need help quickly - enzyme dilution (Jan/05/2010 )
Is the below correct?
I use 0.15ul of Taq polymerase in a 10ul PCR reaction. The Taq stock is sold as 1000U, 5U/ul.
So the final concentration of Taq is (0.15/10)*1000 = 15U
If I have second stock sold as 250U, 5U/ul and want the final concentration in my PCR to be the same as the above, I should add in (x/10)*250 = 15U; x = 0.6ul?
If you are adding 0.15 ul of a 5 U/ul solution, you are adding .75 units. The total volume in your tube is completely irrelevant.
phage434 on Jan 5 2010, 07:36 PM said:
Yes, you are using 0.75 units. If you really want a final concentration then it would be 0.075 units/ul (for a 10ul reaction volume)
Ok, but what I really wanted to know was how many ul to add if I am using a 250U stock versus a 1000U stock? Yes, they both read 5U/ul, but isn't one at a lower stock (the 250U) so I would have to add more of it in a 10ul PCR reaction so the final concentration of the enzyme is the same as if I was pulling from the 1000U stock?
Or does it not matter because they both say 5U/ul? Can someone explain why?
You have two enzyme stocks with the same concentration (5U/ul) therefore you just use the same amount of each for one reaction.
Concentration of your enzyme= total amount of enzyme/ total volume in tube
The 1000U or 250U refers to the total amount of enzyme in your stock tube, and has been taken into account already when calculating the concentration, so you can disregard it.
Think of it like this, if it helps-
Your 250U stock is simply a 1000U stock that has been aliquoted into 4 tubes..........
One time you bought a tube containing 50 ul of a 5 U/ul solution, and another time you bought (probably for around four times as much) one with 200 ul of a 5 U/ul solution. Put them both in the same tube, and you'll have 250 ul of a 5 U/ul solution -- The volume in the tube doesn't matter, the concentration is the same.