Biomedical Engineering Reference
In-Depth Information
Read the absorbances of samples and standards at 600 nm.
1
µ
=
µ
g deoxyribose
6
g DNA.
References
Burton K (1956) Biochem J 62:315
1.2.4 Quantitative DNA Determination
with Fluorescent Dyes
For DNA estimations ethidium bromide (EtBr, excitation wave-
length
λ
=
λ
=
254
or
366 nm,
emission
wavelength
Em
m
Ex
λ
=
500 − 590 nm), 4
,6-diamino-2-phenylindole-2 (DAPI,
372 nm,
Ex
λ
=
λ
=
λ
=
454 nm), or Hoechst 33 258 (
360 nm,
450 nm)
Em
Ex
Em
are suitable.
A E ff : 0mM Tr i s , 1 mM EDTA,madetopH8.0withHCl
Solutions/Reagents
(0.61 g Tris and 0.186 g Na
2
-EDTA
·
2H
2
O in 500 ml ddH
2
O,
aliquoted and autoclaved)
B1
µ
g/ml ethidium bromide in TE buffer by dilution of a stock
solution of 1 mg
/
ml EtBr in Soln. A. (The other mentioned
dyes are used the same way.)
Standard
DNA from calf thymus or phages: 0, 1, 2, 5, 5, 7, 5, 10,
and 20
µ
g DNA per ml TE buffer, stable at −20
◦
C for several
month.
Mix 4
µ
µ
µ
l
of each mixture onto a plastic foil (e.g., Parafilm), lying on a UV
transilluminator. Photograph the spots and evaluate the picture
densitometrically. The optical densities of the standards are plot-
ted against the amount of their DNA and the DNA content of the
samples is calculated from the obtained standard curve (range:
0-15
l of standards and samples with 4
l of Soln. B. Spot 5
µ
g DNA/ml).
The use of PicoGreen for double-stranded DNA (
λ
=
485 nm,
Ex
λ
=
/
µ
/
520 nm, linear range 25 pg
ml to 1
g
ml) and OliGreen
Em
λ
=
λ
=
/
µ
/
(
ml)for
oligonucleotides and single-stranded DNA increases the sensitivity
up to three orders of magnitude
5
. Using these fluorescent dyes,
standards and samples may be measured in a fluorescence microtest
plate reader.
480 nm,
520 nm, linear range 100 pg
ml to 1
g
E
Em
References
Daxhelet GA, Coene MM, Hoet PP, Cocito CG (1989) Anal Biochem 179:401
Ausubel FM et al. (eds.) (1994) Current protocols in molecular biology.
Wiley, New York, Vol. I, 2.6.7-2.6.8
5
Haugland RP (1996) Handbook of fluorescent probes and research
chemicals, 6th ed. pp. 161-164. Molecular Probes, Eugene, Ore-
gon. Technical Bulletin OliGreen ssDNA reagent, and kit (2005), and
PicoGreen reagent and kit (2005), http:// www.invitrogen.com/probes/
