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#1
12-01-2011, 06:30 PM
 Unregistered Guest Posts: n/a
dilution help

I need help with this problem. You don't need to know the science, it is more of a dilution and concentration math problem.

I have a standard reaction setup below with these reagents:

H20 = 32.75ul
10x PCR buffer = 5ul ---> final 1x
MgCl2 (25mM) = 3ul -----> final 1.5mM
Primer F (10uM) = 2.5ul ---> final 0.5uM
Primer R (10uM) = 2.5ul -----> final 0.5uM
dNTP (10mM) = 1ul ------>final 0.2mM
DMSO (100%) = 1ul ------>final 2%
DNA = x
Taq polymerase (5U) = 0.25ul ----> final 0.025U
Total = 50ul

The DNA must be 1ug in 50ul. I have DNA at 20ng/ul.

The only way can get 1ug in 50ul is if I add 50ul of DNA into the reaction. But even if I make H2O = 0ul, I will still be about 15ul over my required 50ul, and the final concentrations of each reagent will change.

I need to have 1ug of DNA in the reaction but keep my final reagent concentrations the same (or close as possible) to the standard reaction above. I am sure I can find a way if I keep plugging away with the numbers but I don't have that much time. Can someone help me?
#2
12-02-2011, 08:41 AM
 Mrs X can't count, can't spell! Join Date: Feb 2006 Location: New Zealand Posts: 2,436 Rep Power: 12
Re: dilution help

Quote:
 Originally Posted by Unregistered I need help with this problem. You don't need to know the science, it is more of a dilution and concentration math problem. I have a standard reaction setup below with these reagents: H20 = 32.75ul 10x PCR buffer = 5ul ---> final 1x MgCl2 (25mM) = 3ul -----> final 1.5mM Primer F (10uM) = 2.5ul ---> final 0.5uM Primer R (10uM) = 2.5ul -----> final 0.5uM dNTP (10mM) = 1ul ------>final 0.2mM DMSO (100%) = 1ul ------>final 2% DNA = x Taq polymerase (5U) = 0.25ul ----> final 0.025U Total = 50ul The DNA must be 1ug in 50ul. I have DNA at 20ng/ul. The only way can get 1ug in 50ul is if I add 50ul of DNA into the reaction. But even if I make H2O = 0ul, I will still be about 15ul over my required 50ul, and the final concentrations of each reagent will change. I need to have 1ug of DNA in the reaction but keep my final reagent concentrations the same (or close as possible) to the standard reaction above. I am sure I can find a way if I keep plugging away with the numbers but I don't have that much time. Can someone help me?
You can't get around it. 1µg = 1000ng.
In order to get 1µg of DNA, you need 50µL of 20ng/µL solution.

Therefore, you need a more concentrated solution to start with, anything you add to it will just be diluting the DNA concentration.
#3
06-15-2012, 06:39 AM
 Unregistered Guest Posts: n/a
Re: dilution help

Hello,

Sorry not to be of any help to your question. But I am having a slight issue with diluting concentrations. I need to use 2mM dNTP in my PCR reactions at a volume of 5 micro litres per sample. Can anyone help me with how I can obtain a 2mM dNTP concentration??

Abbi
#4
06-16-2012, 12:14 PM
 Mrs X can't count, can't spell! Join Date: Feb 2006 Location: New Zealand Posts: 2,436 Rep Power: 12
Re: dilution help

Quote:
 Originally Posted by Unregistered Hello, Sorry not to be of any help to your question. But I am having a slight issue with diluting concentrations. I need to use 2mM dNTP in my PCR reactions at a volume of 5 micro litres per sample. Can anyone help me with how I can obtain a 2mM dNTP concentration?? Many thanks for your advice. Abbi
Hi Abbi, what concentration do you have to start with? 2mM means 2 millimoles per litre.

5µL will need 10 nanomoles of your dNTP

(1mL has 2µmoles, 1µL has 2nanomoles.)

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