Absorbances for diluted samples - (Jul/01/2011 )
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?
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...
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?
check dilution A3 again...
Are you sure you didnt make a typing error there?
Doesn't it need to be 10µl in 990µl or?
Sorry. Both A3 and A5 are 1000x dilution. Edited =x
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.
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.