Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
* * * - - 1 votes

Primer dilution

Primers dilution qPCR

  • Please log in to reply
7 replies to this topic

#1 Mad Researcher

Mad Researcher

    Newbie !!

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 133 posts
7
Neutral

Posted 20 May 2015 - 10:42 AM

Hello,

 

I received my primers today (in a plate) and the datasheet specified to add 100 ul of water to get a 100 micromolar solution.

 

Now, i want to use a 0.3 micromolar solution in my qPCR so how do i dilute it further. I want a final volume of 25 ul.

 

I could dilute my 100 micromolar solution to 10 or 5 micromolar but then again whenever i have to run a qPCR i have to dilute it again. Any solution how to dilute to get a final conc. of 0.3 or 0.4 micromolar solution to be used with qPCR?

 

Thanks smile.png


Edited by Mad researcher, 20 May 2015 - 11:10 AM.

Cheers,

Mad Researcher

#2 r.rosati

r.rosati

    member

  • Active Members
  • Pip
  • 17 posts
0
Neutral

Posted 20 May 2015 - 12:15 PM

Yes, you can dilute it further if you wish, because depending on how many reactions you have, volumes of a 100 uM stock are a bit too small to pipet comfortably.

 

There's a very easy method to calculate dilution, and it's by doing it in two steps.

First determine the dilution factor: let's say you have your 10 uM diluted stock and you want to dilute it to 0.3 uM, so you want to dilute 10/0.3 = 33.3 times.

Since you want to dilute 33.3 times, and your final volume is 25 ul, then the volume of 10 uM stock you'd need to add is 25/33.3 = 0.75 ul per tube.



#3 Mad Researcher

Mad Researcher

    Newbie !!

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 133 posts
7
Neutral

Posted 20 May 2015 - 12:35 PM

Yes, you can dilute it further if you wish, because depending on how many reactions you have, volumes of a 100 uM stock are a bit too small to pipet comfortably.

 

There's a very easy method to calculate dilution, and it's by doing it in two steps.

First determine the dilution factor: let's say you have your 10 uM diluted stock and you want to dilute it to 0.3 uM, so you want to dilute 10/0.3 = 33.3 times.

Since you want to dilute 33.3 times, and your final volume is 25 ul, then the volume of 10 uM stock you'd need to add is 25/33.3 = 0.75 ul per tube.

Thanks for your quick response :)

 

So first i need to dilute my 100 uM stock to 10 uM and then i dilute it to 0.3 uM. Am i correct in saying this?


Cheers,

Mad Researcher

#4 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 6,005 posts
437
Excellent

Posted 20 May 2015 - 12:50 PM

Typical working stocks for PCR are 10 uM, the final dilution to 0.3 uM (or whatever you are using) is done in the reaction mix.

 

Plate based primer preps are easy - if you have the plate made up to 100 uM as suggested, then you can easily make a dilution plate by getting a new plate, adding 90 ul of water to each well, then adding 10 ul of each 100 uM primer stock to the respective well. If you are confident in your pipettor, you can do this with a multichannel pipette.



#5 Mad Researcher

Mad Researcher

    Newbie !!

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 133 posts
7
Neutral

Posted 20 May 2015 - 12:54 PM

Typical working stocks for PCR are 10 uM, the final dilution to 0.3 uM (or whatever you are using) is done in the reaction mix.

 

Plate based primer preps are easy - if you have the plate made up to 100 uM as suggested, then you can easily make a dilution plate by getting a new plate, adding 90 ul of water to each well, then adding 10 ul of each 100 uM primer stock to the respective well. If you are confident in your pipettor, you can do this with a multichannel pipette.

Thanks bob, But i again have to dilute it to reach my desired concentration of 0.3 uM?


Cheers,

Mad Researcher

#6 Mad Researcher

Mad Researcher

    Newbie !!

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 133 posts
7
Neutral

Posted 20 May 2015 - 01:10 PM

I understood this much.

Now, how to calculate how much uL should i add to my reaction to have 0.3 uM final concentration?


Cheers,

Mad Researcher

#7 r.rosati

r.rosati

    member

  • Active Members
  • Pip
  • 17 posts
0
Neutral

Posted 20 May 2015 - 04:23 PM

Say, if you have a 10 uM stock, then by the calculation I wrote, you would use 0.75 ul of this for a 25-ul reaction.



#8 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,631 posts
271
Excellent

Posted 20 May 2015 - 07:13 PM

I normally keep a "master" stock of a primer at 100 uM, and keep a working stock at 10 uM. The working stock I make up in a 200 ul volume, which makes it easy to thaw when I'm ready to do a PCR. With a 10 uM working stock, you typically add 1-2 ul of primer to a 50 ul PCR reaction. I keep both the master and working solutions in TE, not in water. TE preserves the DNA, even at room temperature, essentially indefinitely (although I still keep things frozen). Some people worry about the effect of the TE on reactions; I'd recommend doing the math -- the effects are essentially negligible.







Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.