33 Identification of Cell Lines
331
1. Run PCR amplification of DNA samples according to the protocol given PC R
for the GeneAmp PCR reagent kit. Each PCR reaction consists of 40 fl.l
reaction mixture containing 360 ng eukaryotic DNA and 5 fl.l primer
(stock concentration 100 pmol/fl.l). Adjust the concentration of MgCl 2
to 3 mM. In the reaction mixture, dUTP is substituted for dTTP to
avoid possible carryover and reamplification of PCR products in subsequent runs, leading to false-positive results. Carryover PCR products containing dUTP can be selectively degraded using the enzyme
uracil N-glycosylase.
2. Include a negative control, consisting of distilled water, in place of the
DNA sample in each run. For further reduction of contamination, it is
recommended that autoclaved cotton plugged pipette tips are used
for dispensing DNA and that the PCR reactions are performed in a
separate room to that in which the DNA was isolated.
3. After 2 min initial denaturation at 95°C, conduct 40 cycles of amplification under the following conditions: denaturation at 95°C for 5 s,
annealing at 40°C for 1 s, and extension at noc for 30 s. Following the
last cycle, a final noc extension is performed for 5 min.
4. Cool the samples to 15°C.
1. Take 10 fl.l from each reaction tube, and load this sample into wells in Electrophoresis
a 2.5% Metaphor gel. Electrophorese the samples at a constant voltage
of 125 V for 1 h in 0.5xTBE buffer.
2. Observe the DNA bands under UV light after staining the gel in 1 fl.g/
ml ethidium bromide solution for 15-30 min.
3. Record the DNA bands photographically using a Polaroid camera and
type 55 positive/negative film.
Results
According to McIntosh et al. (1996), discrimination of cell lines from
different species was possible with any primer set (Fig. 2). In their trial,
some cell lines derived from different species showed the same DNA profiles, but this was explained as cross-contamination or mislabelling of
the cultures used.
Subprotocol 4
RAPD-PCR
Kawai and Mitsuhashi (1997) devised a method of discriminating insect
cell lines by RAPD-PCR with a tenmar single primer.
Materials
- Centrifuge and centrifuge tubes
- Microcentrifuge and micro centrifuge tube (1.5 ml)
Equipment
331
1. Run PCR amplification of DNA samples according to the protocol given PC R
for the GeneAmp PCR reagent kit. Each PCR reaction consists of 40 fl.l
reaction mixture containing 360 ng eukaryotic DNA and 5 fl.l primer
(stock concentration 100 pmol/fl.l). Adjust the concentration of MgCl 2
to 3 mM. In the reaction mixture, dUTP is substituted for dTTP to
avoid possible carryover and reamplification of PCR products in subsequent runs, leading to false-positive results. Carryover PCR products containing dUTP can be selectively degraded using the enzyme
uracil N-glycosylase.
2. Include a negative control, consisting of distilled water, in place of the
DNA sample in each run. For further reduction of contamination, it is
recommended that autoclaved cotton plugged pipette tips are used
for dispensing DNA and that the PCR reactions are performed in a
separate room to that in which the DNA was isolated.
3. After 2 min initial denaturation at 95°C, conduct 40 cycles of amplification under the following conditions: denaturation at 95°C for 5 s,
annealing at 40°C for 1 s, and extension at noc for 30 s. Following the
last cycle, a final noc extension is performed for 5 min.
4. Cool the samples to 15°C.
1. Take 10 fl.l from each reaction tube, and load this sample into wells in Electrophoresis
a 2.5% Metaphor gel. Electrophorese the samples at a constant voltage
of 125 V for 1 h in 0.5xTBE buffer.
2. Observe the DNA bands under UV light after staining the gel in 1 fl.g/
ml ethidium bromide solution for 15-30 min.
3. Record the DNA bands photographically using a Polaroid camera and
type 55 positive/negative film.
Results
According to McIntosh et al. (1996), discrimination of cell lines from
different species was possible with any primer set (Fig. 2). In their trial,
some cell lines derived from different species showed the same DNA profiles, but this was explained as cross-contamination or mislabelling of
the cultures used.
Subprotocol 4
RAPD-PCR
Kawai and Mitsuhashi (1997) devised a method of discriminating insect
cell lines by RAPD-PCR with a tenmar single primer.
Materials
- Centrifuge and centrifuge tubes
- Microcentrifuge and micro centrifuge tube (1.5 ml)
Equipment