Bio-Rad DCode™ Universal Mutation Detection System - Manual (Page 5)

Section 4
Troubleshooting
Refer to the DCode universal mutation detection system manual for
more troubleshooting details.
4.1 PCR
Problem Cause Solution
Faint, visible bands Unused primers 1. A 19 bp fragment is not a prob-
lem.
Concatemer DNA 2. Amplified DNA forms concate-
mer size DNA. This is not a
problem when the sample is
denatured for the SSCP gel.
No 299 bp fragment No DNA 1. Make sure DNA is added to
PCR reaction.
Inactive enzyme 2. Use viable Taq DNA poly-
merase.
Missing or bad dNTPs 3. Make sure all four dNTPs are
used in PCR reaction.
Missing primer/primers 4. Make sure both primers are
used in PCR reaction.
Numerous bands Nonspecific priming 1. Do set-up in PCR designated
area.
2. Make sure not to cross contami-
nate the mutant and wild-type
DNA.
7
Fig. 1. Separation of mutant and wild-type DNA on the DCode sys-
tem. The samples were electrophoresed on an 8% acrylamide/bis gel
containing 7% glycerol for 3.5 hours at 30 W with the buffer chilled at
10 °C. The gel was stained with SyBr Green II. Lane 1 contains the
mutant DNA and lane 2 contains the wild-type DNA.
6
Single strand DNA
Single strand DNA
Double strand DNA
1 2
4100103d.qxd 11/4/98 8:48 AM Page 6