Bio-Rad DCode™ Universal Mutation Detection System - Manual (Page 57)

4. Crosslinking ratio: The acrylamide/bis ratio determines the percent of crosslinking. SSCP
gels generally use 1–2 % crosslinking. Acrylamide concentrations will vary from 5% to 10%.
5. Buffer concentration: Gels are run with TBE buffer at concentrations of 0.5x or 1.0x. In
some cases, 0.5x TBE appears to give slightly better results than 1.0x TBE. 23
SSCP protocols have typically used radioisotope-labeled fragments, but recently nonra-
dioactive or “cold SSCP” methods have been developed. 24 For a more complete description
of the SSCP technique, refer to references 1, 22, and 25–29.
When connected to an appropriate external chiller, the DCode system can control the
buffer temperatures between 5–25°C. The electrophoresis cooling tank is outfitted with two
cooling fingers. Tygon tubing connects the cooling fingers in the electrophoresis tank to an
external chiller. The chiller recirculates a coolant through the cooling fingers which, in turn,
cools the buffer. The external chiller is set to chill the coolant to approximately –20°C, and the
DCode heater regulates the buffer temperature. An example of an SSCP gel run on the DCode
system is shown in Figure 6.1.
1 2 3 4
Fig. 6.1. Amplified mutant and wild-type alleles of exon 8 from the p53 gene. Separation by SSCP run at constant
30 W for 3.5 hours in 1x TBE on an 8% acrylamide gel (37.5:1) with 3.5% glycerol at 8°C. Lane 1, undenatured mutant allele;
lane 2, mutant allele; lane 3, wild-type allele; lane 4, undenatured wild-type allele.
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