Notes, Buffers, And Volumes - Hoefer HE33 User Manual

Notes, buffers, and volumes

Agarose gel electrophoresis notes
Agarose gel electrophoresis can be used to
separate DNA fragments as small as 0.1 kb or
less. Polyacrylamide gels are usually used for
fragments smaller than 1 kb.
DNA mobility
Suggested agarose concentration for sepa-
rating fragments of various sizes is given in
Table 2 below. Other factors affecting separa-
tion results include the running buffer, volt-
age setting, temperature, conformation, and
the presence of ethidium bromide. Special
agaroses are available that can extend resolu-
tion ranges.
A common standard is a Hind III digest of
lambda phage, which gives eight fragments
ranging in size from 0.1 to 23 kb pairs. For
good resolution, run 45 minutes on a 10-cm
long, 1% agarose gel in 0.5X TBE gel
at 150 V.
Table 2:
Agarose concentrations for separating DNA
fragments of various sizes
agarose (%)
0.5
0.7
1.0
1.2
1.5
*Current Protocols in Molecular Biology, p 2.5.2 (1993)
effective range of resolution
of linear DNA fragments* (kb)
1.0–30
0.8–12
0.5–10
0.4–7
0.2–3
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