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
- - - - -

Resuspending primers calculation


  • Please log in to reply
4 replies to this topic

#1 Paty

Paty

    member

  • Active Members
  • Pip
  • 5 posts
2
Neutral

Posted 11 July 2013 - 06:55 PM

I'm having problems trying to understand de chemestry in resuspending primers. I know I can use online calculators, and I have the general formula like multiply the initial (dry) concentration x10 and get a final concentration of 100 ul/L or

"convert from nmole to umole then divide by the desired concentration in umole/L. The umoles cancel out giving the needed volume in liters. We then convert liters to ml."
Still my question is the initial concentration wich comes in nm is nm/L or nm/?? Because molarity is always expressed for a volume of solution. Then when we divide we are not dividing only nm but nm/some volume

Please help I'm an anthropologist so this part of work is hard for me. Sorry for my english!
Thank you in advance,
Patricia


#2 pcrman

pcrman

    Epigenetist

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,165 posts
68
Excellent

Posted 11 July 2013 - 08:37 PM

Usually primers (oligos) come in lyphilized powder with the amount (mass) indicated on the tube, for example, 4.9 nmol. We usually reconstitute the primer to make a 100 uM/L stock solution, we will need to add 49 ul TE buffer to the tube (4.9 nM/49 ul = 100 um/L). So if you know the amount of the primer from the tube, it is easy to determine how much solvent is needed without any calculation.

To put it in a simple way, if you have x nmol dry weight primer, you add 10 * x ul TE buffer, you will obtain 100 uM/L solution.

#3 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,811 posts
136
Excellent

Posted 12 July 2013 - 03:43 AM

be careful with nomenclature. uM=umol/L. uM/L is, at best, redundant, at worst, meaningless.
talent does what it can
genius does what it must
i do what i get paid to do

#4 Paty

Paty

    member

  • Active Members
  • Pip
  • 5 posts
2
Neutral

Posted 19 August 2013 - 03:48 PM

Thank you very much for your answers. My confusion is with nomenclature indeed. 



#5 memari

memari

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 148 posts
11
Good

Posted 20 August 2013 - 06:23 AM

We multiply nmol of primers by 50 to find the micro liter of water that should be added to them.

 

for example:

primer GAPDH-F  nMol is  20

so multiply it by 50=>  20*50= 1000

now add  1000 microlitter of clean water to it and mix.

now 1micro litter is enough for 10 to 50 micro litter of  pcr reaction.


Edited by memari, 20 August 2013 - 06:26 AM.

-----
Babak Memari




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

©1999-2013 Protocol Online, All rights reserved.