- Load 2–5X the amount of DNA that would give bands of moderate intensity
on an ethidium bromide-stained gel. Typically, this is something on the
order of 0.5–2.5 µg of a 1-kb fragment on a 30 mL 1% mini gel. These
numbers are guesstimates so your mileage may vary.
- Run the gel normally and then place it in a 0.002% methylene blue
(w/v, Sigma M-4159) solution in 0.1X TAE (0.004M Tris 0.0001 M EDTA)
for 1–4 hrs at room temperature (22°C) or overnight at 4°C. Diffusion of the
DNA does not seem to be a problem for fragments as small as 100 bp (3%
Nusieve:1% agarose gel). This avoids background issues associated with
staining with 0.02% methylene blue for 30–60 min and then destaining for
a long time.
- If destaining is needed to increase the visibility of the bands, place the gel
in 0.1X TAE with gentle agitation, changing the buffer every 30–60 min
until you are satisfied with the degree of destaining.
This method primarily eliminates the damage of DNA by UV irradiation. DNA
isolated from MB stained gels should transform frozen competant E. coli (XL1-
Blue and DH5) cells on the order of 20–50-fold more efficiently than EB-isolated
DNA. Factors influencing improved efficiency are: time factor (degradation, etc.),
transilluminator wavelength and intensity, and the %AT of your DNA to mention
a few. One of the advantages of MB staining is the elimination of several of the
Both FMC GTG agarose and Nusieve GTG perform very well. Synergel is
incompatible with MB (very high background). MB should be compatible with
polyacrylamide (even less of a background problem).
Nuieve:Agarose (3:1, 4% final) gels stain very nicely and dsDNA as small
as 75 bp is easily visualized.
Recovery of DNA from Low-melting-temperature Agarose Gels
Caution: Ultraviolet radiation and EtBr in a gel are dangerous. Wear protective
goggles and gloves to protect the eyes and EtBr contamination.
- Digest the plasmid-DNA (up to 20 µg)-containing insert.
- Pour a gel containing the appropriate concentration of low-meltingtemperature
- Mix the samples of DNA with tracking dye, heat shock at 65°C for 5 min,
transfer in ice, and load onto the gel.
- Carry out electrophoresis at 12 volts overnight.
DNA of a given size runs slightly faster through gels cast with lowmelting-
temperature agarose than through conventional gels.
- Take a picture.
- Using a handheld UV light with a long wavelength (to minimize the
damage to the DNA), cut out insert bands using a scalpel. Cut the gel as
close to band of interest as possible and transfer it to a clean 1.5 mL MFT.
Check or draw the removing band on the picture for a record of which
band was eluted.
- Add ≈ 5 volumes of H2O to the slice of agarose.
- Melt the agarose at 65°C for 1 to 5 min. Vortex for 20 sec and store at
–20°C. Further Separate the DNA from the Agarose.
- Spin the tubes (4K, 10 min, 20°C).
- Transfer the supernatant with a micropipette (P-20) into new MFT. The
white substance at the interphase is powdered agarose.
- Re-extract the agarose phase once with phenol:chloroform and once with
- Transfer the aqueous phase to a MFT, bring up to 105 mL with H2O, add
35 µL NH4Ac and 1050 µL EtOH, and invert to mix.
- Centrifuge 5 K, 20 min at 4°C.
- Take supernatant, rains with 70% EtOH, dry and resuspended in 5–10 µL
TE. And store at –20°C.