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# How to dilute the template DNA for PCR

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### #1 chemgirl02

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Posted 10 April 2012 - 11:50 AM

Someone recommend to me to dilute the DNA template, but Im getting a little lost in the math. My original template DNA is 1300ng/uL and I want the concentrations to be in 10ng, 25ng, and 50ng. I will be doing a 50uL PCR rxn. Thanks for the help in advance.

### #2 pito

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Posted 11 April 2012 - 01:08 AM

You want 10ng in total (so 10 ng in 50µl) or you want 10ng per 1µl and thus 500ng in 50µl ?

Does C1 x V1 = C1 x V1 says anything to you?

And think about this: you have 1300ng per 1 µl, so how many µl do you need to take to have 1 ng?
1300ng in µliter mean that you have to dilute it, since its more then the 10ng in total (or 50ng) for 50µl ...
But first you need to know what it will be: 10ng in 50µl or 500ng in 50µl and then we can help.
But think about it yourself, if you have 1300ng per µl, how can you then dilute it?
ex given: you need 13ng in 50µl , how would you do this then?
I would start with: 1300 ng per µl, I take 1 µl and have this 1300ng , I add 9 µl water, meaning I diluted 1/10 , so I have in the end 10µl with 130ng in it...so whats next...
If you don't know it, then ask it! Better to ask and look foolish to some then not ask and stay stupid.

### #3 chemgirl02

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Posted 11 April 2012 - 07:23 PM

You want 10ng in total (so 10 ng in 50µl) or you want 10ng per 1µl and thus 500ng in 50µl ?

Does C1 x V1 = C1 x V1 says anything to you?

And think about this: you have 1300ng per 1 µl, so how many µl do you need to take to have 1 ng?
1300ng in µliter mean that you have to dilute it, since its more then the 10ng in total (or 50ng) for 50µl ...
But first you need to know what it will be: 10ng in 50µl or 500ng in 50µl and then we can help.
But think about it yourself, if you have 1300ng per µl, how can you then dilute it?
ex given: you need 13ng in 50µl , how would you do this then?
I would start with: 1300 ng per µl, I take 1 µl and have this 1300ng , I add 9 µl water, meaning I diluted 1/10 , so I have in the end 10µl with 130ng in it...so whats next...

It's 10ng in 50uL total.

### #4 chemgirl02

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Posted 11 April 2012 - 07:33 PM

You want 10ng in total (so 10 ng in 50µl) or you want 10ng per 1µl and thus 500ng in 50µl ?

Does C1 x V1 = C1 x V1 says anything to you?

And think about this: you have 1300ng per 1 µl, so how many µl do you need to take to have 1 ng?
1300ng in µliter mean that you have to dilute it, since its more then the 10ng in total (or 50ng) for 50µl ...
But first you need to know what it will be: 10ng in 50µl or 500ng in 50µl and then we can help.
But think about it yourself, if you have 1300ng per µl, how can you then dilute it?
ex given: you need 13ng in 50µl , how would you do this then?
I would start with: 1300 ng per µl, I take 1 µl and have this 1300ng , I add 9 µl water, meaning I diluted 1/10 , so I have in the end 10µl with 130ng in it...so whats next...

From your example I think I answered my own question but please correct me if I am wrong.

For 10ng in 50uL with a starting DNA at 1300ng/uL

(1300) x X = (10ng)(50uL)--->1300 x X = (500)--->500/1300= x
x= 0.38ul added to 49.62uL of H2O

For the 25ng in 50uL with a starting DNA at 1300ng/uL

(1300) x X =(25ng) (50uL)--->1300 x X =(1250)--->1250/1300=x
x=0.96uL added to 49.04uL of H2O

For 50ng in 50uL with a starting DNA at 1300ng/uL

(1300) x X =(50uL) (50ng)---> 1300 x X= (2500)---> 2500/1300=x
x=1.92uL added to 48.08uL of H2O.

Is this correct???

### #5 pito

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Posted 11 April 2012 - 11:57 PM

You want 10ng in total (so 10 ng in 50µl) or you want 10ng per 1µl and thus 500ng in 50µl ?

Does C1 x V1 = C2 x V2 says anything to you?

And think about this: you have 1300ng per 1 µl, so how many µl do you need to take to have 1 ng?
1300ng in µliter mean that you have to dilute it, since its more then the 10ng in total (or 50ng) for 50µl ...
But first you need to know what it will be: 10ng in 50µl or 500ng in 50µl and then we can help.
But think about it yourself, if you have 1300ng per µl, how can you then dilute it?
ex given: you need 13ng in 50µl , how would you do this then?
I would start with: 1300 ng per µl, I take 1 µl and have this 1300ng , I add 9 µl water, meaning I diluted 1/10 , so I have in the end 10µl with 130ng in it...so whats next...

From your example I think I answered my own question but please correct me if I am wrong.

For 10ng in 50uL with a starting DNA at 1300ng/uL

(1300) x X = (10ng)(50uL)--->1300 x X = (500)--->500/1300= x
x= 0.38ul added to 49.62uL of H2O

For the 25ng in 50uL with a starting DNA at 1300ng/uL

(1300) x X =(25ng) (50uL)--->1300 x X =(1250)--->1250/1300=x
x=0.96uL added to 49.04uL of H2O

For 50ng in 50uL with a starting DNA at 1300ng/uL

(1300) x X =(50uL) (50ng)---> 1300 x X= (2500)---> 2500/1300=x
x=1.92uL added to 48.08uL of H2O.

Is this correct???

Redo the math.

If you take 0.38ul from the 1300ng/µl, then how much ng do you have? => 1300 x 0,38 = 494 ng of DNA and you would add this in a total volume of 50µl, meaning 494ng/50µl = 9,88ng/µl.... So do you think this is ok?
Its not... if you want 10ng in total!

Are you sure you want 10ng in 50µl or 10ng/µl and thus 500ng(494) in total?

Its its the first, you are not right.

ANd the reason why is because you do not use a concentration in your formula..

Its C x V = C x V
and the first C, you use 1300ng/µl , which is ok (but write down the ng/µl, because not doing it, would result in mistakes.. one you are making allready now)
the first V you use ? , its ok , its what you want to know
the second C you use 10ng... but what is this? 10ng? Thats not a concentration, its either 10ng/µl (as it is now, the way its written now, you use 10ng/µl) or 10ng/50µl (meaning 0,2ng/µl)
and the second V should be 50 µl then.

So....
If you don't know it, then ask it! Better to ask and look foolish to some then not ask and stay stupid.

### #6 GNANA

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Posted 12 April 2012 - 12:56 AM

Hi chem.....,
Think pito has given enough input to make you understand the math involved in your calculation, anyways i just give you the solution in other way. first do a 1:100 dilution of your template (1 micro litr of temp+99 micro litres of H2O), this will give you 13ng/micro litre of template conc. from which you got to take 0.77 micro litre (10/13) for 10ng PCR, 1.92 micro litre (25/13) for 25 ng PCR, and 3.84 micro litres (50/13) for 50 ng PCR.....hope this helps....

Gnana....
I would prefer being perfectionist rather than a passionist in Research.

I always had an alternate hypothesis....

### #7 chemgirl02

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Posted 12 April 2012 - 01:53 PM

Hi chem.....,
Think pito has given enough input to make you understand the math involved in your calculation, anyways i just give you the solution in other way. first do a 1:100 dilution of your template (1 micro litr of temp+99 micro litres of H2O), this will give you 13ng/micro litre of template conc. from which you got to take 0.77 micro litre (10/13) for 10ng PCR, 1.92 micro litre (25/13) for 25 ng PCR, and 3.84 micro litres (50/13) for 50 ng PCR.....hope this helps....

Gnana....

Okay I was wondering about the concentration of the 10ng.

Okay so if I go Pito's example of diltuing my template to a more reasonable amount which is 1:100 dilution giving me 13ng/uL.

I can solve the 10ng/50uL

13ng/uL x X = (10ng/50uL) x 50uL---> (0.2ng/uL x 50uL)/13ng/uL=x---> (10ng)/(13ng/uL)=0.77uL I need to add.

KK this makes more sense. When I was going from the 1300ng/uL I was getting such a small number I thought I was doing the math completely wrong. Thanks again!

### #8 pito

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Posted 12 April 2012 - 02:05 PM

Hi chem.....,
Think pito has given enough input to make you understand the math involved in your calculation, anyways i just give you the solution in other way. first do a 1:100 dilution of your template (1 micro litr of temp+99 micro litres of H2O), this will give you 13ng/micro litre of template conc. from which you got to take 0.77 micro litre (10/13) for 10ng PCR, 1.92 micro litre (25/13) for 25 ng PCR, and 3.84 micro litres (50/13) for 50 ng PCR.....hope this helps....

Gnana....

Okay I was wondering about the concentration of the 10ng.

Okay so if I go Pito's example of diltuing my template to a more reasonable amount which is 1:100 dilution giving me 13ng/uL.

I can solve the 10ng/50uL

13ng/uL x X = (10ng/50uL) x 50uL---> (0.2ng/uL x 50uL)/13ng/uL=x---> (10ng)/(13ng/uL)=0.77uL I need to add.

KK this makes more sense. When I was going from the 1300ng/uL I was getting such a small number I thought I was doing the math completely wrong. Thanks again!

Thats what I allready mentioned in my first post...
because you have such high amount to start with.. you would indeed need to take a very very small amount (not realistic) so yeah, you need to dilute first..
If you don't know it, then ask it! Better to ask and look foolish to some then not ask and stay stupid.