Eletrophoresis tips

DGGE METHOD

http://www-odp.tamu.edu/publications/187_SR/203/203_3.htm

Western Blot Protocol

http://labs.fhcrc.org/fero/Protocols/western.html

Eletrophoresis

Eletrophoresis

• When preparing agarose for electrophoresis, it is best to sprinkle the agarose into room-temperature buffer, swirl, and let sit at least 1 min before microwaving. This allows the agarose to hydrate first, which minimizes foaming during heating.
• Electrophoresis buffer can affect the resolution of DNA. TAE (Tris-Acetate-EDTA) buffer provides better resolution of fragments >4 kb, while TBE (Tris-Borate-EDTA) buffer provides better resolution of 0.1- to 3-kb fragments. In addition, use TBE buffer when electrophoresing >150 V and use TAE buffer with supercoiled DNA for best results.
• Migration of DNA is retarded and band distortion can occur when too much buffer covers the gel. The slower migration results from a reduced voltage gradient across the gel..
• Loading DNA in the smallest volume possible will result in sharper bands.
• You can preserve DNA in agarose gels for long-term storage using 70% ethanol. [See Jacobs, D. and Neilan, B.A. (1995) BioTechniques 19, 892.]
• Electrophoresing a gel too “hot” can cause the DNA to denature in the gel. It can also cause the agarose gel to deform. Cool the gel with a small fan during the electrophoresis.
• For the Supercoiled DNA Ladder electrophoresed on <1% agarose gels, add 2 1lg/ml ethidium bromide to the gel. Otherwise, smeared bands and extra bands will be seen because of different degrees of supercoiling. [See Longo, M.C. and Hartley, J.L. (1986) Focus 8:3, 3. (reprinted on page 63, this issue).]
• When glycerol-containing loading buffers are used in DNA samples electrophoresed through acrylamide gels, smiling bands may be accentuated especially in TBE.
• On a polyacrylamide gel, DNA fragments having AT-rich regions migrate slower than other DNA fragments of the same size. This anomalous migration is enhanced at lower temperatures and disappears at high temperatures. This anomalous migration is not observed on agarose gels. [See Stellwagen, N.C. (1983) Biochemistry22, 6186.]
• The minimum amount of DNA detectable by ethidium bromide on a 3-mm-thick gel and a 5-mm-wide lane is 1 ng. Do not exceed 50 ng of DNA per band on a 3-mm-thick gel and 5-mm-wide lane.

Web source: http://www.bio.davidson.edu/courses/molbio/tips/funDNAgel.html