94
KAI-OLAF NETZER
I OB. Remove the 20 g needle. Insert a suitable comb 10 mm deep. Allow one
hour for polymerization at room temperature.
IIB. Preheat approximately 5 1 of 1x TAE buffer (for Hoefer Scientific
SE600) to 60°C in a microwave oven.
Note: The buffer can be reused 3 times.
12B. Preheat the water bath to approximately 70°C. Connect the external
circulation with the heat exchanger of the electrophoresis unit. Fill the
buffer tank with preheated buffer.
13B. Place the buffer tank on the magnetic stirrer. Stir at medium velocity.
Insert a thermometer into the tank and adjust the buffer temperature
to 60 °C.
14B. Carefully remove the comb from the gel. Disassemble the casting apparatus and insert the gel into the tank. Flush the wells thoroughly
with running buffer using a syringe and a 20 gauge needle.
Note: Wait 10 min for temperature equilibration before loading the gel.
ISB. Add 1/5 volume gelloading buffer to each DNA sample (approximately 100 ng in 10 J.tl, 1/10 of a 50 J.tl PCR reaction). Load the samples
into the wells of the gel.
16B. Run the gel for approximately 500 Vh (see "Optimizing Conditions").
Recirculate the buffer using a peristaltic pump (10 ml/min).
17B. Disassemble the apparatus and stain the gel (see "Staining").
Comments
Optimizing conditions
Gradient range To find optimal conditions, it is advisable to first analyze a PCR product in a
perpendicular gel with a wide gradient range, i. e. 0-80%. This helps to elucidate the melting temperature of domains within the DNA fragment. The
curve obtained for a molecule with one domain accessible for DGGE will be
of sigmoid shape: High electrophoretic mobility in the region oflow denaturant concentration, reduced mobility in the region of high denaturant
concentration. A point of inflection in the curve (see Figure 2) occurs at
the denaturant concentration that is equivalent to the "melting temperature" of a melting domain (i. e., the denaturant concentration at which 50%
ofthe melting domain is denatured and 50% is non-denatured). The "melt-
KAI-OLAF NETZER
I OB. Remove the 20 g needle. Insert a suitable comb 10 mm deep. Allow one
hour for polymerization at room temperature.
IIB. Preheat approximately 5 1 of 1x TAE buffer (for Hoefer Scientific
SE600) to 60°C in a microwave oven.
Note: The buffer can be reused 3 times.
12B. Preheat the water bath to approximately 70°C. Connect the external
circulation with the heat exchanger of the electrophoresis unit. Fill the
buffer tank with preheated buffer.
13B. Place the buffer tank on the magnetic stirrer. Stir at medium velocity.
Insert a thermometer into the tank and adjust the buffer temperature
to 60 °C.
14B. Carefully remove the comb from the gel. Disassemble the casting apparatus and insert the gel into the tank. Flush the wells thoroughly
with running buffer using a syringe and a 20 gauge needle.
Note: Wait 10 min for temperature equilibration before loading the gel.
ISB. Add 1/5 volume gelloading buffer to each DNA sample (approximately 100 ng in 10 J.tl, 1/10 of a 50 J.tl PCR reaction). Load the samples
into the wells of the gel.
16B. Run the gel for approximately 500 Vh (see "Optimizing Conditions").
Recirculate the buffer using a peristaltic pump (10 ml/min).
17B. Disassemble the apparatus and stain the gel (see "Staining").
Comments
Optimizing conditions
Gradient range To find optimal conditions, it is advisable to first analyze a PCR product in a
perpendicular gel with a wide gradient range, i. e. 0-80%. This helps to elucidate the melting temperature of domains within the DNA fragment. The
curve obtained for a molecule with one domain accessible for DGGE will be
of sigmoid shape: High electrophoretic mobility in the region oflow denaturant concentration, reduced mobility in the region of high denaturant
concentration. A point of inflection in the curve (see Figure 2) occurs at
the denaturant concentration that is equivalent to the "melting temperature" of a melting domain (i. e., the denaturant concentration at which 50%
ofthe melting domain is denatured and 50% is non-denatured). The "melt-