Bio-Rad DCode™ Universal Mutation Detection System - Manual (Page 3)

Section 2
PCR Reaction
Note: It is extremely important that solutions and materials used during
PCR set-up are not exposed to amplified DNA to avoid contamination
during amplification.
1. Add the components listed below to 200 µl or 500 µl thin-walled
microfuge tubes and mix.
Tube 1 Tube 2
Wild-type DNA Mutant DNA
Control DNA 1.5 µl 1.5 µl
Primer A 2.5 µl 2.5 µl
Primer B 2.5 µl 2.5 µl
10x Taq polymerase buffer 10.0 µl 10.0 µl
2.5 mM dNTPs 8.0 µl 8.0 µl
Taq DNA polymerase enzyme (5 U/µl) 0.5 µl 0.5 µl
Sterile water 75.0 µl 75.0 µl
Total volume 100.0 µl 100.0 µl
Note: 10x Taq polymerase buffer contains 100 mM Tris-HCl pH 8.3,
500 mM KCl, 15 mM MgCl
2
, and 0.01% gelatin. If desired, add a
third tube which contains no control DNA as a negative control.
2. Place the tubes into a thermocycler and enter the following parameters.
Step 1 94 °C 2 minutes x 1 cycle
Step 2 94 °C 45 seconds
61 °C 45 seconds x 35 cycles
72 °C 45 seconds
Step 3 72 °C 10 minutes x 1 cycle
3. To check the product, add 5 µl of the amplified DNA to gel loading
buffer and run on a 10% acrylamide/bis gel (10% Ready Gel—cata-
log number 161-0905). Run a DNA size standard, such as Bio-Rad’s
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Sequence of Primer A: 5’- GGG CTG GGC ATA AAA GTC A -3’
Sequence of Primer B: 5’- AAT AGA CCA ATA GGC AGA G -3’
2x SSCP Gel Loading Dye: 95% Formamide, 20 mM EDTA pH 8.0,
0.05% Xylene Cyanol, and 0.05%
Bromophenol Blue
1.2 Additional Supplies Required
Taq DNA polymerase enzyme
10x Taq polymerase buffer
2.5 mM dNTPs
Sterile water
Thin-walled microfuge tubes - 200 µl or 500 µl size
Sterile aerosol tips
1.3 Storage Conditions
All kit components should be stored at -20 °C. The shelf life of the
kit stored at -20 °C is 1 year.
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