Bio-Rad DCode™ Universal Mutation Detection System - Manual (Page 2)

Section 1
Introduction
The DCode control kit for SSCP (Single Stranded Conformational
Polymorphism) provides reagents that are used to prepare mutant and
wild-type DNA for gel electrophoresis with the DCode universal mutation
detection system. The SSCP technique is based on the fact that single-
stranded DNA has a defined secondary structure. Sequence differences
as small as a single base change can affect this secondary structure and
can be detected by electrophoresis in a nondenaturing polyacrylamide
gel.
1 Double stranded mutant and wild-type samples are first denatured
into single strands and then loaded onto the gel. Differences in mobili-
ty of the single strands between the wild-type DNA and the other sam-
ples indicate a mutation. SSCP is a widely used mutation screening
method because of its simplicity.
The DCode control kit for SSCP contains wild-type DNA, mutant
DNA, two 19 base-pair oligonucleotide primers, and 2x SSCP gel loading
dye. The mutant DNA is identical to the wild-type DNA, except for a
single A to T mutation at fragment base number 110. The PCR reaction
will produce a 299 bp fragment. There are sufficient amounts of DNA
and primers for five 100 µl PCR reactions for both the mutant and wild-
type DNA.
1.1 Kit Components
Item Concentration Amount Volume
Mutant DNA 50 pg/µl 500 pg 10 µl
Wild-type DNA 50 pg/µl 500 pg 10 µl
Primer A 10 pmol/µl 400 pmol 40 µl
Primer B 10 pmol/µl 400 pmol 40 µl
2x SSCP Gel Loading Dye 2x 1 ml
1
Table of Contents
Section 1 Introduction................................................................... 1
1.1 Kit Components ......................................................................... 1
1.2 Additional Supplies Required.................................................... 2
1.3 Storage Conditions..................................................................... 2
Section 2 PCR* Reaction .............................................................. 3
Section 3 SSCP Gel ....................................................................... 4
Section 4 Troubleshooting ............................................................ 7
4.1 PCR............................................................................................ 7
4.2 SSCP Gel ................................................................................... 8
Section 5 Reference ....................................................................... 8
* The Polymerase Chain Reaction (PCR) process is covered by patents owned by Hoffman-
La Roche.
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