Biomedical Engineering Reference
In-Depth Information
C 50% glycerol (v/v), 0.3% bromophenol blue (w/v) in buffer A
D1mg
/
ml ethidium bromide
13
in ddH
2
O
Caution!
Wear gloves! Ethidium bromide is carcinogenic.
The agarose (Soln. B) is molten in a boiling water bath and cooled to
about 50
◦
C. Soln. D is added to a final concentration of 0.5
µ
/
ml
14
.
The liquid agarose is poured into the gel chamber of a horizontal
electrophoresis apparatus and the comb is inserted.
The sample is dissolved in a buffer with low ionic strength and
mixed with 1
g
/
5 of its volume of Soln. C. The amount of DNA should
µ
be about 50
g per square centimeter of slot area.
Electrodebufferis1:5dilutedbufferA,adjustedtopH8.2and
supplemented with D to a final concentration of 0.5
µ
/
ml.
Electrophoresis is performed with 20 - 60 V cv overnight and
the applied voltage is inverse proportional to the mean DNA size.
The addition of ethidium bromide (EtBr) or propidium iodide
(PI) allows the identification of DNA bands immediately after run
on a transilluminator. Intercalation of the dyes into the double-
stranded DNA yields fluorescence (EtBr: excitation wavelength
g
λ
ex
λ
λ
302 or 366 nm, emission wavelength
em
590 nm;PI:
ex
530 nm,
λ
em
620 nm).
Separated DNA may be eluted from cut pieces of gel or trans-
ferred to membranes by Southern blot.
References
Rickwood D, Hames BD (eds.) (1984) Gel electrophoresis of nucleic acids:
a practical approach. IRL Press, Oxford
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory
manual, 2nd ed., vol. 1, chap. 6, Cold Spring Harbor Laboratories Press
Jany K-D, Hahn H (1991) In: Bertram S, Gassen G (eds.) Gentechnische
Methoden. pp. 39-43. Fischer, Stuttgart
2.2.2 Denaturating Nucleic Acid Electrophoresis
General differences between DNA and RNA in denaturating elec-
trophoresis do not exist; therefore, the following protocols may be
used,aswellasforDNAandRNA.
A 0mM sodium phosphate, pH 6.5 - 6.8
Solutions/Reagents
A
10 mM sodium phosphate, pH 7.4, 1.1 M formaldehyde
13
Instead of EtBr use less harmfull fluorescent dyes, e.g., propidium iodide
(PI) or SYBR Green (cf. Haugland RP (1996) Handbook of fluorescent
probes and research chemicals, 6th ed., Chap. 8, Molecular Probes,
Eugene, Oregon; https://catalog.invitrogen.com)
14
If ethidium bromide is omitted as well as in gel and electrode buffer,
theDNAsampleissupplementedwith5
µ
l of Soln. D per milliliter and
incubated for 10 min at RT.
