Dilution, Cell concentration & DNA content - (Oct/05/2011 )
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.
In the plates, there was gradual decrease in colonies from one dilution to the next.
I need help.
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?
1) possibly - need more details
2)Beer's law (Beer-Lambert law)
3) Maybe - limits of the kit?
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.
Thanks bob and phage.
I suspected that the relationship established earlier might be wrong and is trying to make a new calibration graph.