Polymerase chain reaction amplification products separated on rehydratable polyacrylamide gels and stained with silver.
Separation of polymerase chain reaction (PCR) amplification of specific fragment length polymorphisms was carried out on rehydratable polyacrylamide gels on a horizontal flat slab system. A discontinuous sulfate-borate buffer system was employed on 5-8% T gels crosslinked with 3.5% C. Samples were diluted in leading sulfate ion buffer at 1/10 the ionic strength of the separating gel buffer and placed directly onto the surface of the rehydrated gels in 0.5-10 microliters volumes. The trailing ion and counterion were contained in a gel plug and placed directly onto the anodal and cathodal ends of the gel, and the electrodes placed directly onto the surface of the gel plugs. Filter paper wicks, soaked in diluted leading ion buffer, were placed along each side to lower the ionic strength of the edges, thereby increasing mobility at the edge and thus preventing smile effects. The gel-gel contact of the plug and separating gel prevent the production of a junction potential which occurs between dissimilar materials such as a paper wick and the gel. Ten- to 20-cm separations were carried out from 2-5 h, respectively, and resolution in the 20 cm system was 1.6-4 bp (base pairs) between 100 and 500 bp, 4-7 bp between 500 and 1000 bp, 12-20 bp between 1000 and 2000 bp and about 50 bp between 2000 and 3000 bp. Between 3000 and 4000 bp, resolution fell off to +/- 100 bp. Sensitivity, using a silver stain, indicated that one could readily distinguish less than 10 pg of DNA per mm width on the gels.(ABSTRACT TRUNCATED AT 250 WORDS)
Allen
R C
RC
Dept. of Pathology and Laboratory Medicine, Medical University of South Carolina, Children's Hospital, Charleston 29425.
Graves
G
G
Budowle
B
B
D001709:Biotechnology; D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D012834:Silver; D013194:Staining and Labeling; D014867:Water
D014867:Water; D012834:Silver; D004247:DNA