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

Detection of Nucleic Acids 73
on ice before loading. Add 1 µg of radioactively labeled DNA molecular weight marker to
1 µL of gel loading buffer in one lane.
5. Electrophorese at 30 mA for 2 to 3 h or at constant temperature (see Note 20).
6. Remove gel from the electrophoresis tank and discard radioactive buffer.
7. Place gel plates on tissue paper and insert a scalpel between the back plate and spacers.
Remove the backplate trying to ensure that the gel remains on the front plate (see Note
21).
8. Immerse gel, on plate, in gel fixation solution and fix for 15 to 30 min. Carefully remove
the gel, on the plate, from the fixative, drain, and cover with a dry piece of Whatman
3MM filter paper.
9. Either invert the gel and carefully lift the glass plate away from the paper or lift the paper
gently from the glass plate. The gel should stick to the paper.
10. Dry on a gel dryer for 60 min at 80°C under vacuum (see Note 22).
11. Cover gel with clingfilm and place autoradiography film directly over the gel and expose
at –70°C (see Note 23).
4.2.3. Recovery of DNA from Acrylamide Gels
Recovery of DNA from acrylamide is a relatively simple procedure because of the
higher amounts of DNA that can be loaded on acrylamide gels, with yields of around
50 to 60% sufficient DNA recovered for most applications. We have only applied this
technique to unfixed gels stained with ethidium bromide.
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
acrylamide present in the gel slice, the better the recovery.
2. Transfer to a microcentrifuge tube and add 400 µL of Maxam & Gilberts solution.
2. Vortex and incubate overnight at 37°C.
3. Spin down the gel by pulse centrifugation for 30 s at 5000g in a bench-top centrifuge.
4. Remove supernatant to a separate Eppendorf tube (approx 350 µL).
5. Add 1 µL of 10 mg/mL tRNA (optional, omit if required).
6. Add 2.5 volumes of ice-cold 100% ethanol and mix gently.
7. Stand for 20 min and centrifuge for 10 min at 15,000g.
8. Decant supernatant and add 300 µL of 70% ethanol, mix gently.
9. Centrifuge for 10 min at 15,000g.
10. Decant supernatant and air dry pellet.
11. Resuspend in 20 µL of distilled water.
5. Notes
1. For some applications, lower concentrations of dyes can give better results, for example,
when the DNA fragment runs close to one of the dyes. In this case, dilute loading buffer
15 in 30% glycerol.
2. Ethidium bromide is a carcinogen, teratogen, and mutagen. Handle with respect.
3. Digesting plasmids with enzymes such as HinfI leaves 5′ overhangs that can be labeled
with Klenow using the following reaction: 1 µg of digested plasmid in 5 µL of distilled
water, 2 µL of 10 × repair buffer supplied with Klenow); 1 µL of a mixture containing
2 mM each of dGTP, dCTP, and dTTP; 2 µL of 35 S-dATP (approx 0.5 µCi), and 1 µL of
Klenow fragment of DNA polymerase (5–10 units) in a total volume of 20 µL. Incubate
for 30 min at room temperature and stop reaction by adding 2 µL of 0.25 M EDTA. Store
at –20°C and use 0.5 to 1 µL per gel.