some body to help me in my primer dilution?

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MolBio
MolBio's picture
some body to help me in my primer dilution?

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

ARGERINE
ARGERINE's picture
Dear Molbio

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

Ivan
Ivan's picture
 

 
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
newcommers's picture
Dear ARGERINE

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
newcommers's picture
Dear IVAN

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
Ivan's picture
I am not sure I understand

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
newcommers's picture
Dear IVAN

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
Ivan's picture
 

 
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
newcommers's picture
Dear IVAN

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
Ivan's picture
 

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

harry348
harry348's picture
 Can anyone tell me how to

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

ARGERINE
ARGERINE's picture
Dear harry

Dear harry

dilute your sample 2x. 

Ivan
Ivan's picture
Argerine is correct. Just mix

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
RSHENEE's picture
THANKS IVAN AND ARGERINE...

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
newb's picture
I know this might be stupid,

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

Ivan
Ivan's picture
Hi newb,

Hi newb,

This is not a stupid question, just the type of question asked in a test. 

Good luck working out the answer.

newb
newb's picture
I swear I'm not taking a test

I swear I'm not taking a test on this :). I'm an intern at PNNL and I think you just do (112µL * 100pmol)/(10pmol) right?
 

Ivan
Ivan's picture
To make a 1:10 dilution, all

To make a 1:10 dilution, all you do is add one part of your stock solution into 9 parts of water (hence the term "one in ten" dilution = one part in a total of 10 parts). 

For example, for a final volume of 112 ul, you need to add 11.2 ul of your stock solution into 100.8 ul of water. 11.2 is one part in ten out of a total of 112. Another way to look at it is: divide your desired volume, 112, by 10, and the answer, 11.2, is the one part in ten you need. 

newb
newb's picture
Oh my lanta, really? Thanks

Oh my lanta, really? Thanks Ivan, and I feel very stupid, and hopefully I can get better at this.

Ivan
Ivan's picture
No worries. The math is easy,

No worries. The math is easy, the trick is knowing what you are supposed to be doing and that comes with experience. When it comes to primers most of the time you will be working with stock solutions (100 pmol/ul) and working stocks (10 pmol/ul), so you will be preparing those solutions regularly.

By the way, 100 pmol/ul is the same as 100 uM (micromolar).

Good luck.

Chinnaraja
Chinnaraja's picture
Dear IVAN

Dear IVAN
I have one doubt in my PCR preparations. I prepared 10uM primer concentration as a working stock. In that i have no doubt. But if i take 1ul from that 10uM working stock for 25ul PCR reaction what is a concentration of primer in that 1ul primer? is it 100nmol? Pls clarify soon.

Ivan
Ivan's picture
If you add 1 ul of a 10 uM

If you add 1 ul of a 10 uM stock into a 25 ul reaction, then the concentration of your primer, in the 25 ul reaction, would be 1/25th of 10 uM, or 0.4 uM (which is the same as saying 400 nM). 

Cheers

Chinnaraja
Chinnaraja's picture
Dean IVAN,

Dean IVAN,

Thank you so much... Its really helpful...longtime doubt cleared now...Cheers :-)

Chinnaraja
Chinnaraja's picture
Dear Ivan

Dear Ivan
If i need 40 nmol or 40pmol concentration of primer from 10uM primer stock, then howmuch i supposed to take from 10uM working stock...

Ivan
Ivan's picture
Chinnaraja,

Chinnaraja,

I strongly recommend that you read a little about how to make these calculations. This is basic math and something you have to know as a scientist. The calculation I gave you in the earlier message is all you need to know, and it should be straight forward for you to use the example I gave you to make the calculations you need. 

Good luck. 

Chinnaraja
Chinnaraja's picture
k thank you...:-). no worries

k thank you...:-). no worries

nazi ihsun
nazi ihsun's picture
 its very simple to make 40pm

 its very simple to make 40pm concentration from 10um primer stock.
if 1 ul has 10pm conc then taking 4 ul wil have 40pm concentration
example;
10um=10pm/1ul means 1 ul has 10pm
1ul=10pm
 x   =40pm by cross multiplication
x=4ul