Negative staining with zinc-imidazole of gel electrophoresis-separated nucleic acids.
Zinc and imidazole salts were applied for the detection of nucleic acids on either polyacrylamide of agarose gels. After electrophoresis, polyacrylamide gels are washed in distilled water to remove most of the residual electrophoresis reagents, then incubated in 10 mM zinc sulfate for 10 min, and subsequently immersed in 0.2 M imidazole for 3 min. As a result, zinc salts precipitate on the gel surface, except in the positions occupied by nucleic acids, which appear as transparent, colorless bands. Staining of nucleic acids on agarose gels can be performed by incubation in 40 mM zinc sulfate for 10 min, followed by immersion in 0.2 M imidazole for 5 min to form a deep white-stained background. On soaking in 2 M imidazole for 45 min, the imidazole-induced zinc precipitate is removed from the positions were nucleic acids are located resulting in a negative image of colorless and transparent nucleic acid bands against a white background. The sensitivity of this stain ranges from 5 to 7 ng/band for small (from 1 to 0.2 kbp) DNA, from 7.8 to 13 ng/band for different 22-base oligonucleotides, from 62 to 125 ng/band for large (from 20 to 2 kbp) DNA, and is 1 microgram/band for human peripheral-blood monocyte RNA. After chelation of zinc with EDTA, the nucleic acids can be quantitatively recovered from the gel. The principal advantage of this technique over ethidium bromide staining is evident for preparative purposes. Using zinc-imidazole in the detection of purified pBACIB.1 (2.8 kbp) plasmid DNA and anti-HBsAg single chain Fv antibody fragment (0.7 kbp) DNA, followed by elution from gel slices, ligation and transformation of competent E. coli XL-1 Blue cells, the number of transformants notably increased from 280 (obtained with conventional ethidium bromide staining plus UV-irradiation at 312 nm) to 10,000.
Hardy
E
E
Center for Genetic Engineering and Biotechnology, Havana City, Cuba.
Pupo
E
E
Casalvilla
R
R
Sosa
A E
AE
Trujillo
L E
LE
López
E
E
Castellanos-Serra
L
L
D000914:Antibodies, Viral / Q000235:genetics; D011232:Chemical Precipitation; D004247:DNA / Q000302:isolation & purification / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004492:Edetic Acid; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004996:Ethidium; D006514:Hepatitis B Surface Antigens / Q000276:immunology; D006801:Humans; D007093:Imidazoles; D008970:Molecular Weight; D009696:Nucleic Acids / Q000302:isolation & purification*; D010957:Plasmids; D013194:Staining and Labeling; D055672:Static Electricity; D015032:Zinc / Q000378:metabolism; D019287:Zinc Sulfate
D000914:Antibodies, Viral; D006514:Hepatitis B Surface Antigens; D007093:Imidazoles; D009696:Nucleic Acids; D019287:Zinc Sulfate; C029899:imidazole; D004247:DNA; D004492:Edetic Acid; D004262:DNA Restriction Enzymes; D004996:Ethidium; D015032:Zinc