90
KAI-OLAF NETZER
70-400 bp long. 1J.1g/gel is required forperpendicular DGGE (i.e. product
from a 100 Jll PCR reaction), 50-100 ng/lane for parallel DGGE (i.e. approximately 1/10 of a 50 Jll PCR reaction). Crude PCR reactions can be
used, if no non-specific product is visible on agarose gels.
Procedure
ln advance 1. Prepare the DNA fragment by PCR using the appropriate conditions. At
the conclusion of 35 cycles, perform a 5 minutes/72 oc step to fill in single-stranded tails.
2. Cast a 1.8% agarose gel to check each PCR reaction for the presence of
products of correct length and absence of non-specific products.
3. If mutation screening is intended, mix halfof a patient's reaction mixture
with the same amount of normal control mixture using a fresh 0.5 ml
PCR tube. The remaining half of the patients' mixture is run in a parallel
line. Alternatively, a mixture from a different patient may be substituted
for control if distinct mutations are expected. Overlay with 50 Jll mineral
oil and place the tubein a temperature cycler. Use the following program
for denaturing/reannealing to generate heteroduplex molecules: 95°C, 10
min; 55°C, 1 h, with a temperature slope of 0.1 °C/sec in between.
4. Prepare two DGG stock solutions containing 0%, and 80% denaturing
agents ( 100% corresponds to 40% formamide/7 M urea):
Component
Amount to give 100 mL
0%
80%
Urea
33.6 g
Formamide
32 ml
AcrylamideiBis 38.4: 1.6
17 ml
17 ml
SOx T AE buffer
2 ml
2 ml
Ultrapure water
81 ml
21,7 ml
Store protected from light at 4°C. Solutions should be used within two
weeks.