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dilution help

pcr

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#1 molbio1234

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Posted 01 December 2011 - 07:28 PM

I have a standard 1x PCR reaction setup below:

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 1x 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 leelee

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Posted 01 December 2011 - 07:38 PM

Why do you need to have 1ug of DNA in your reaction? That seems a lot of DNA to me....

If I were you, I would add 1ul of your template and run the PCR and just see how it goes.

#3 molbio1234

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Posted 01 December 2011 - 07:57 PM

I was told to use 1ug of DNA by my boss. I am sure there is a reason for it.

Edited by molbio1234, 01 December 2011 - 08:01 PM.


#4 leelee

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Posted 01 December 2011 - 08:35 PM

What DNA is your template? (e.g. plasmid? genomic?)

Also:
Maybe you should go and ask your boss about it then?
If he/she has a reason for it, you should probably know what it is.

Or, if you can't ask them for whatever reason, is there someone else in your lab who can help you with PCR?

#5 Trof

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Posted 02 December 2011 - 01:36 AM

I agree that 1ug is really much of template.
I personally hate being told what to do without knowing why (or getting someones plasmid to cut and don't know sequence, do a reaction for someone and don't know what they want to find out,...) and since some time back I refuse to do anything on such conditions, it doesn't pay back.

If you really have a reason for using 1 ug it's better to concentrate your DNA if you have enough of it (because anyway 20ng/ul is a low concentration) either by vacuum concentrator or precipitation with ethanol (but that only if you have enough of overal yield of DNA as precipitation doesn't work well will small overal amounts).

If you can't do that, you can resort to increasing the reaction volume to 72 ul. That way you multiply everything by 1.44. (but your setup you wrote doesn't add up to 50, only 48ul, so you have to count with water 34.75ul, not 32.75). The final concentrations will remain the same. Except of course the concentration of the template, but unless you concentrate you can't move it.

Edited by Trof, 02 December 2011 - 01:42 AM.

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#6 molbio1234

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Posted 02 December 2011 - 05:46 AM

Hi Trof

Thank you for the help. How did you figure out to multiply by 1.44?

#7 Trof

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Posted 02 December 2011 - 06:11 AM

You said you had 32.75 of H2O in your reaction that you can substitute with DNA (actually 34.75). And you need 50ul to have 1ug. So I calculated which overal reaction volume is proportionate to using at least 50ul of H2O. That is (50/34.75)/50 = 71,94.... So round off to 72 and that is 144% of your original volume.

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon






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