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
- - - - -

Absorbances for diluted samples


  • Please log in to reply
6 replies to this topic

#1 Ic3_88

Ic3_88

    member

  • Active Members
  • Pip
  • 19 posts
5
Neutral

Posted 01 July 2011 - 08:39 AM

I got a 10x, 100x and 10000x diluted sample. My result obtain was the more diluted the sample is, the more concentration of the DNA. Could anyone kindly explain how reliable are the absorbances for more dilute samples?

#2 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,800 posts
407
Excellent

Posted 02 July 2011 - 02:20 PM

Can you show us the calculations you performed to get the reults, and if possible, the spectrograph.

You should also think about sensitivity of the assay (working range) and possibly other things that can interfere with the assay...

#3 Ic3_88

Ic3_88

    member

  • Active Members
  • Pip
  • 19 posts
5
Neutral

Posted 02 July 2011 - 11:38 PM

We are given 5 tubes (A1,A2,A3,A4 and A5).

A1 = 100ul DNA + 900ul H20
A2 = 100ul from A1 + 900ul H20
A3 = 100ul from A2 +900ul H20
A4 = 10ul DNA + 990ul of H20
A5 = 10ul from A4 + 990ul of H2O

The DNA provided was not really pure. Below is the calculation of dilution factor I have done and the OD260,OD280 and OD260/OD280

** Assuming one A260 unit equals to 60ug of double stranded DNA/ml

Dilution Factor OD 260 OD280 OD260/OD280 Concentration of Stock (OD260 x 50 x Dilution factor)
A1 10, 2.408, 2.659, 0.9058, 1204ug/ml
A2 100, 0.444, 0.307, 1.4452, 2220ug/ml
A3 1000, 0.044, 0.048, 0.9260, 22000ug/ml
A4 100, 0.542, 0.353, 1.5364, 2710ug/ml
A5 1000, 0.008, 0.029, 0.2833, 4000ug/ml



The questions are:
1) How reliable are the absorbances for more dilute sample
2) The tubes in A2 and A4 are of the same dilution factors, why are the concentration different?

Edited by Ic3_88, 03 July 2011 - 09:02 AM.


#4 pito

pito

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,334 posts
81
Excellent

Posted 03 July 2011 - 04:09 AM

check dilution A3 again...
Are you sure you didnt make a typing error there?

Doesn't it need to be 10Ál in 990Ál or?

Edited by pito, 03 July 2011 - 04:09 AM.

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#5 Ic3_88

Ic3_88

    member

  • Active Members
  • Pip
  • 19 posts
5
Neutral

Posted 03 July 2011 - 09:01 AM

Sorry. Both A3 and A5 are 1000x dilution. Edited =x

#6 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,800 posts
407
Excellent

Posted 03 July 2011 - 03:46 PM

So your numbers are telling me that your DNA is very impure - do you know how I can tell this? From that you can have a guess at what might be going on to give you higher concentrations at bigger dilutions...

If you know how the DNA was prepared you can probably even go one step futher and guess what the contaminant is.

#7 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,805 posts
133
Excellent

Posted 13 July 2011 - 06:36 AM

you should also take into consideration the absorbance values. in the old days you wanted to have your readings within the range of 0.1-0.7 absorbance units. in more modern spectrophotometers the useful range was expanded to 0.1-1.0 absorbance units. you have to question the validity of readings outside this range.
talent does what it can
genius does what it must
i do what i get paid to do




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

©1999-2013 Protocol Online, All rights reserved.