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
- - - - -

Primer Concentration Help


  • Please log in to reply
4 replies to this topic

#1 cjmills

cjmills

    member

  • Members
  • Pip
  • 3 posts
0
Neutral

Posted 16 June 2010 - 07:08 AM

Hi everyone,

I am trying to optimize a multiplex PCR and am missing a band. I want to try to run my control with just 1 pair of primers and am confused as to how to calculate the volume required. My rxn volume is 25 ul, the final concentration of each primer is listed as 6.25 pmol. The initial concentration of my primers is 12.5 uM. My "go to" equation is (C1)(V1) = (C2)(V2), but I don't know how to used this when C1 is in uM and C2 is in pmole (not pM). Can anyone help me? This is driving me crazy. Thank you!

#2 Trof

Trof

    Brain on a stick

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,199 posts
109
Excellent

Posted 16 June 2010 - 07:52 AM

You have to make clear if you need a concentration - uM, pM (means umol/L, pmol/L) or amount (umol, pmol only).

If there is supposed to be 6.25 pmol in 25 ul reaction, that means final concentration is 6.25/25 = 0.25 pmol/ul = 0.25 umol/L = 0.25 uM.

You can then use the equation, I think.

I count it differently. You need 6.25 pmols in reaction (which is by the way not much, I usualy use 10 pmols), in 1 ul of your stock solution there is 12.5 pmols (12.5 uM = 12.5 umol/L = 12.5 pmol/ul) so:

1 ul .......12.5
x ul ........6.25
x = 0.5 ul

So you need 0.5 ul of each primer

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


#3 cjmills

cjmills

    member

  • Members
  • Pip
  • 3 posts
0
Neutral

Posted 16 June 2010 - 08:17 AM

Thank you so much for your reply, Trof. I thought there were 10^6 pmole in 1 umole? Shouldn't the final concentration be 0.0025 uM?

That is what I keep getting and it seems like a ridiculously low number.

Thanks,
Carrie

#4 Trof

Trof

    Brain on a stick

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,199 posts
109
Excellent

Posted 17 June 2010 - 02:07 AM

Thank you so much for your reply, Trof. I thought there were 10^6 pmole in 1 umole? Shouldn't the final concentration be 0.0025 uM?

There is, but there's 10^6 ul in liter. So pmol/ul equals umol/L.

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


#5 cjmills

cjmills

    member

  • Members
  • Pip
  • 3 posts
0
Neutral

Posted 17 June 2010 - 09:37 AM

Duh! Thanks Trof! I could not figure out what I was doing wrong. ;)




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

©1999-2013 Protocol Online, All rights reserved.