Sensitivities In Comparison; Results From Plasmid And Blood Dna Sample - tecan GENios Operating Manual

9. Application Note for DNA Quantification
9.3.2

Sensitivities in comparison:

9.3.3

Results from Plasmid and Blood DNA Sample

(a) DNA from Blood
Treatment
485/535
(b) Plasmid DNA
485/535 nm
Abs 260 nm
9-4 Operating Manual for GENios, GENios FL, and GENios Plus No: I 112 904 Rev No: 1.1
260 nm absorbance:
PicoGreen
fluorescence:
The comparison between absorbance and fluorescence measurements show that
fluorescence quantitation is 100 to 1000 times more sensitive. The calibration
curves show the linear range of the different methods. It should be emphasized,
that the sensitivities are dependent on filling volumes (especially for absorbance),
flash numbers, type of diluent and even the type of DNA can influence the final
result.
In some cases the amount of blood and DNA is not sufficient for UV quantitation.
DNA from human blood was extracted with different treatments. The volume and
concentration of the extracted samples did not allow to quantify with absorbance.
1 10 µl blood fresh/EDTA
2 20 µl blood fresh/EDTA
3 10 µl blood fresh/Citrate
4 20 µl blood fresh/Critrate
1
1.02 1.06
Table 1 Calculated DNA concentrations in µg/ml stock solution of the same
samples with different treatment.
The same samples from a plasmid extraction have been measured with both
methods. Since the fluorescence method is a lot more sensitive, the samples
were diluted 1:100 before the measurement – both of the detection techniques
show comparable results.
46 44 46
44 45 45
Table 2 Comparison of the stock concentrations (referring to the calibration
curves above) in µg/ml of the samples measured with fluorescence (485/535) and
absorbance (260).
approximately 50 – 250 ng DNA/ml
approximately 300 pg DNA /ml
5
6
7
8
2 3 4
2.99 4.15
49 59 50 39
65 53 46 36
10 µl blood old/EDTA
20 µl blood old/EDTA
10 µl blood old/Citrate
20 µl blood old/Critrate
5 6 7
3.88 3.92 1.23
48 48 51 43
49 38 46 55
8
2.27
48 36
54 34
June 2002