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# some body to help me in my primer dilution?

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MolBio
Israel

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Status: Frog Egg
 Topic Started by MolBio on 6/14/2009 9:58 AM

I have a big problem because I don't now how I need to do a dilution primer....

The liophilizated concentration primer is 32.2 nmol and the concentratios is 100 uM the report say :

to 100 pM/ul dissolve in (ul) 320 ul of DDW...I need to keep the 0.36 uM work solution. I used the formule to calculate Cnewx  Vnew =  Cstock x  V stock  and If i calculate right I need to add 115 to 1000ul is correct what I say or I wronged in my calcule?

If some body help me please I very very appreciate...

Thanks

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ARGERINE
India

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Status: Cancer, Diagnostics & Proteomics Moderator
 Posted By ARGERINE on 6/14/2009 12:53 PM

Dear Molbio

I will give you an illustrative example so that you can do it on your own

First dissolve fresh primer with the 10X volume as its concentration:
For eg., for 25 nmoles of primer add 250 ul of water
This will give you a concentration of 0.0001 M or 0.1 mM

(25 nmoles/L) (x moles/250 x 10-6 L) = .0001 mole/L = 0.0001 M = 0.1 mM

To prepare primer for use:
Dilute this stock 1:10, to give a concentration of 10 mM

From this, use 1 ml in a typical PCR reaction. This will give you a final concentration of 10 pmoles in a PCR reaction.
(Because 1 mL of 10 mM = 10 mmoles/10-6 L = 0.00001 mmole/ml = 10 pmol/ml)

I hope that will work for you

Gaganjot Singh Truth seems so closer now......

Ivan
United States

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Status: DNA Moderator
 Posted By Ivan on 6/15/2009 10:12 AM

Hi MolBio,

Let's see if I understand your situation. You have a 100 uM stock of your primer (32.2 nmols dissolved in 320 ul of water) and you are trying to make a 0.36 uM solution (stock). Your calculations lead you to believe that if you take 115 ul of your 100 uM stock and mix it with 885 ul of water (for a total of 1,000 ul), you will get the 0.36 uM solution you are looking for. Unfortunately this is on the case.

In order to make a 0.36 uM stock you need to mix 3.6 ul of your 100 uM stock with 999.64 ul of water.

Now why you would want to make such a low concentration primer solution is not really clear to me. If you are running a standard PCR reaction, all you need to do is dilute your 100 uM stock 1:10 to make a 10 uM stock. Then when you prepare your PCR reaction you just add enough of your 10 uM primer solution to your PCR reaction such that the final primer concentration is 1 uM (a 1:10 dilution).

Hope this helps.

newcommers
United States

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Status: Frog Egg
 Posted By newcommers on 3/8/2010 12:51 PM

Dear ARGERINE
i understand how to make permament solution (100uM) but still confuse to make 10mL working stock. Would you mind to explain how to make 10mL working stock. Should we 1mL from original primer that we received from vender or that permanent stock that we made recently? Thank you.
newcommers

newcommers
United States

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Status: Frog Egg
 Posted By newcommers on 3/8/2010 12:53 PM

Dear IVAN
i understand how to make permament solution (100uM) but still confuse to make 10mL working stock. Would you mind to explain how to make 10mL working stock. Should we 1mL from original primer that we received from vender or that permanent stock that we made recently? Thank you.
newcommers

Ivan
United States

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Status: DNA Moderator
 Posted By Ivan on 3/8/2010 13:19 PM

I am not sure I understand what you are asking. What do you mean when you say "10mL working stock". Is that 10 milliliters of a 10 micromolar solution? What is the concentration of the primer you received from your supplier?

newcommers
United States

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Status: Frog Egg
 Posted By newcommers on 3/8/2010 13:50 PM

Dear IVAN
Thank you so much for your quick response. I have 47.0 nMole from suplier and now i prepared 100 uM stock by adding 470 ul water. Now, i want to make 10 uM working solution (1:10). As i read your reply to MolBio which was "all you need to do is dilute your 100 uM stock 1:10 to make a 10 uM stock". So, i have a problem to make a 10 uM stock. Thank you.
newcommers

Ivan
United States

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Status: DNA Moderator
 Posted By Ivan on 3/8/2010 13:53 PM

All you need to do to make a 10 uM stock from a 100 uM stock is dilute 1:10. For example, add 100 uL of your 100 uM stock to 900 uL of water and you will get a 10 uM stock. Easy :)

newcommers
United States

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Status: Frog Egg
 Posted By newcommers on 3/8/2010 14:15 PM

Dear IVAN
Thank you so much. I really appreciate. Is that a same water that we use earlier (for 100 uM stock). Thank you.
newcommers

Ivan
United States

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Status: DNA Moderator
 Posted By Ivan on 3/8/2010 14:21 PM

Yes, you can use the same water you used to resuspend your 100 uM stock.

harry348
Netherlands

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Status: Frog Egg
 Posted By harry348 on 5/24/2011 5:24 AM

Can anyone tell me how to dilute 100uM stock primer concentration to 50uM???

ARGERINE
India

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Status: Cancer, Diagnostics & Proteomics Moderator
 Posted By ARGERINE on 5/24/2011 11:51 AM

Dear harry

Gaganjot Singh Truth seems so closer now......

Ivan
United States

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Status: DNA Moderator
 Posted By Ivan on 5/24/2011 11:54 AM

Argerine is correct. Just mix equal volumes of your 100 uM stock and water. For example mix 50 uL of your 100 uM stock with 50 uL of water. This will give you 100 uL of a 50 uM stock.

RSHENEE
Malaysia

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Status: Frog Egg
 Posted By RSHENEE on 11/18/2011 5:01 AM

THANKS IVAN AND ARGERINE... THE EXPLANATION U GAVE WAS AWESOME... I WAS BROWSING SO MANY WEBSITES JUST TO UNDERSTAND THIS CALCULATIONS.. BUT I ONLY UNDERSTOOD FROM THE EXPLANATION GIVEN HERE... THANK U SO MUCH :)

newb
United States

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Status: Frog Egg
 Posted By newb on 7/19/2012 8:19 AM

I know this might be stupid, but using these 5 volumes:

112 µL

372 µL

529 µL

777 µL

936 µL

prepare a 1:10 dilution of your 100 pmol/µL stock primer (final working concentration 10 pmol/µL) into each of the above volumes.

How do I do that???
newb

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