TY - JOUR
T1 - Fast and sensitive silver staining of DNA in polyacrylamide gels
AU - Bassam, Brant J.
AU - Caetano-Anollés, Gustavo
AU - Gresshoff, Peter M.
N1 - Funding Information:
This work was supported by an endowment to the Racheff Chair Excellence in Plant Molecular Genetics, University of Tennessee. The first two authors made an equal contribution to this work.
PY - 1991/7
Y1 - 1991/7
N2 - The photochemically derived silver stain of nucleic acids in polyacrylamide gels originally described by Merril et al. (1981, Science 211, 1437-1438) was modified to reduce unspecific background staining and increase sensitivity (down to 1 pg/mm2 band cross-section). Detection limits for double-stranded DNA fragments from HaeIII endonuclease digests of phage φX174 were maintained despite eliminating oxidation pretreatment of fixed gels and reducing silver nitrate concentration. Preexposure to formaldehyde during silver impregnation enhanced sensitivity and the inclusion of the silver-complexing agent sodium thiosulphate in the image developer decreased background staining. Higher formaldehyde concentration during image development resulted in darker bands with good contrast. The procedure almost halves the number of steps, solutions and experimental time required and can be used for the staining of DNA fragments in polyacrylamide gels bound to a polyester backing film by controlling temperature during image development. We have applied this improved staining procedure for the routine analysis of complex DNA profiles generated by DNA amplification fingerprinting (DAF).
AB - The photochemically derived silver stain of nucleic acids in polyacrylamide gels originally described by Merril et al. (1981, Science 211, 1437-1438) was modified to reduce unspecific background staining and increase sensitivity (down to 1 pg/mm2 band cross-section). Detection limits for double-stranded DNA fragments from HaeIII endonuclease digests of phage φX174 were maintained despite eliminating oxidation pretreatment of fixed gels and reducing silver nitrate concentration. Preexposure to formaldehyde during silver impregnation enhanced sensitivity and the inclusion of the silver-complexing agent sodium thiosulphate in the image developer decreased background staining. Higher formaldehyde concentration during image development resulted in darker bands with good contrast. The procedure almost halves the number of steps, solutions and experimental time required and can be used for the staining of DNA fragments in polyacrylamide gels bound to a polyester backing film by controlling temperature during image development. We have applied this improved staining procedure for the routine analysis of complex DNA profiles generated by DNA amplification fingerprinting (DAF).
UR - http://www.scopus.com/inward/record.url?scp=0025835296&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0025835296&partnerID=8YFLogxK
U2 - 10.1016/0003-2697(91)90120-I
DO - 10.1016/0003-2697(91)90120-I
M3 - Article
C2 - 1716076
AN - SCOPUS:0025835296
SN - 0003-2697
VL - 196
SP - 80
EP - 83
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 1
ER -