John M. S. Bartlett.pdf - Bio-Nica.info

Detection of Nucleic Acids 71
8. Mix DNA with gel loading buffer by adding 11 DNA solution to gel loading buffer.
Slowly add approx 5 to 10 µL sample per well. Add 1 µg of DNA molecular weight marker
to 4 µL of water and 5 µL of gel loading buffer to one lane (see Note 6).
9. Close the lid of the tank to generate an electrical circuit (see Note 7) and run at a voltage
of 1 to 5 volts/cm. Check bubbles are arising from the anode and that the dye is migrating
into the gel.
10. When migration of markers is complete, remove gel and visualize under ultraviolet light
and photograph.
11. Use intensity of bands to estimate DNA concentration; densitometric scanning of the
photograph can also be used as appropriate.
4.1.1. Staining of Agarose or Acrylamide Gels
1. Remove the gel from the electrophoresis tank and stain in 0.5 to 1 µg/mL ethidium
bromide in sufficient electrophoresis buffer to cover the gel for about 10 to 20 min at
room temperature.
2. Destain in distilled water for 2 × 20 min.
3. Visualize gel (see Note 7).
4.1.2. Decontamination of Ethidium Bromide Solutions
Both electrophoresis buffers from ethidium bromide containing gels and staining/
destaining of gels should be decontaminated before disposal (see Note 2).
1. Add 1 g/L activated charcoal.
2. Stir for 30 to 60 min at room temperature.
3. Remove charcoal by filtration and discard solution.
4. Place charcoal and agarose gel into solid waste for incineration (see Note 8).
4.1.3. Recovery of DNA from Agarose Gels
Recovery of DNA from agarose gels produces highly variable yields dependent on
fragment length. Long fragments (5–10 kb and above) are prone to shearing if vortexed.
We have described here the agarose method that is most widely applicable, in our
hands; however, postrecovery purification with phenol chloroform greatly improves
the purity of the DNA and is recommended where postrecovery enzyme modifications
are planned. The use of TAE buffer is recommended and care should be taken not
to overload the gel.
1. After running the gel, visualize the band of interest under ultraviolet light and excise using
a scalpel (see Note 9). Remove the band as cleanly as possible; the lower the amount of
agar present in the gel slice, the better the recovery.
2. Transfer up to 200 mg of agarose gel slice to a microcentrifuge tube and melt at 75°C
(5–10 min. see Note 10) then cool to 45°C.
3. Estimate volume of melted gel and add 2% 50× agarose buffer and mix gently (see
Note 11).
4. Add β-agarose (add 3–4 units per 100 mg of a 1% agarose gel), mix gently (see Note 11),
and incubate at 45°C for 3 to 4 h (see Note 12).
5. Add an equal volume of phenolchloroform and mix by gentle inversion 10 to 20 times.
6. Centrifuge at 3000g for 5 to 10 min.