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
- - - - -

Dilution, Cell concentration & DNA content

Spectrometry Dilution Cell density DNA extraction

  • Please log in to reply
3 replies to this topic

#1 ImNew

ImNew

    member

  • Active Members
  • Pip
  • 5 posts
0
Neutral

Posted 05 October 2011 - 03:30 AM

Hi all,

From the cell concentration and absorbance relationship determined in earlier stages of my project; I allow the cells to grow to desired concentration.

Eg. Absorbance of 0.5 = ~10^12 cells

After that, serial dilutions were done so that the cells number were 10^11, 10^10.... 10^0. And the dilutions were plated.

However, when I measure the absorbance of the dilutions, they don't corresponds to the relationship determined earlier.

And the DNA content were extracted from the cells of each dilutions by DNeasy blood & tissue kit; their concentration were determined using nanodrop.

But the DNA content didn't change much and remained at almost similar level in all dilutions. Posted Image
In the plates, there was gradual decrease in colonies from one dilution to the next.

I need help.Posted Image

1.) Did I do something wrongly?
2.) Why did the absorbance not corresponds to the relationship determined earlier.
3.) DNA content should decrease as cell number decrease. Right?

Thank you.

Regards,
ImNew.

#2 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,521 posts
371
Excellent

Posted 05 October 2011 - 01:43 PM

1) possibly - need more details
2)Beer's law (Beer-Lambert law)
3) Maybe - limits of the kit?

#3 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,383 posts
227
Excellent

Posted 05 October 2011 - 02:26 PM

You could be overloading your DNA column, so that you have reached the maximum binding capacity of the column at the lower dilutions. You would see the same amount of DNA (the amount binding to the column) even though the cells were diluted. At higher dilutions, you eventually may dilute enough to see lower amounts of DNA binding.

#4 ImNew

ImNew

    member

  • Active Members
  • Pip
  • 5 posts
0
Neutral

Posted 08 October 2011 - 12:27 AM

Thanks bob and phage.

I suspected that the relationship established earlier might be wrong and is trying to make a new calibration graph.





Also tagged with one or more of these keywords: Spectrometry, Dilution, Cell density, DNA extraction

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

©1999-2013 Protocol Online, All rights reserved.