4. Photograph visualized bands under UV light and store digitally
by scanning.
5. Compare the patterns of restriction fragment length polymorphism (RFLP) of the isolates Steinernema or Heterorhabditis to
profiles reported in the literature.
5.9 Electrophoresis
1. Separate the resulting fragments by electrophoresis on a 2%
agarose gel containing ethidium bromide (0.2%) in 0.5Â TBE
buffer (54.0 g Tris base + 27.5 g boric acid + 20 ml 0.5 M
EDTA (pH 8.0) to 1 l aqua test).
2. Visualize and capture photographs of the fragments under UV
light.
3. Compare RFLP patterns with published evidence on described
species [176].
5.10 RAPD
(Randomly Amplified
Polymorphic DNA)
Analysis
RAPD is used for the genetic variability and phylogenetic studies of
diverse organisms for resolving taxonomic problems. RAPD detects
polymorphisms with a sole primer of arbitrary nucleotide sequence.
RAPD has the benefit that no previous information of the genome
is needed for effective application. RAPD approach is a comparatively straightforward technique to apply, and the number of loci
that can be scrutinized is unlimited.
5.10.1 Materials
Required
l
Source: Nematode DNA.
l
Equipment: Spectrophotometer, pH meter, vortex, refrigerator,
electrophoresis unit, PCR, gel documentation.
l
Chemicals: PCR mixture.
l
PCR mixture consists of:
– Taq DNA polymerase: 1.5 U.
– dNTPs (2.5 mM each): 2 μl.
– Taq buffer (10Â): 2.5 μl.
– MgCl 2 : 2 Mm.
– Primer: 0.5 μM.
– Genomic DNA: 100 ng.
– Double distilled to make up the volume 25 μl.
5.10.2 Procedure
1. The nucleotide sequences of RAPD primers are shown in the
Table 1.
2. Carry out the PCR in 25 μl volume containing PCR mixture.
3. Preheat the reaction mixture at 95
C and incubate for 5 min at
94
C.
Molecular Characterization
91
by scanning.
5. Compare the patterns of restriction fragment length polymorphism (RFLP) of the isolates Steinernema or Heterorhabditis to
profiles reported in the literature.
5.9 Electrophoresis
1. Separate the resulting fragments by electrophoresis on a 2%
agarose gel containing ethidium bromide (0.2%) in 0.5Â TBE
buffer (54.0 g Tris base + 27.5 g boric acid + 20 ml 0.5 M
EDTA (pH 8.0) to 1 l aqua test).
2. Visualize and capture photographs of the fragments under UV
light.
3. Compare RFLP patterns with published evidence on described
species [176].
5.10 RAPD
(Randomly Amplified
Polymorphic DNA)
Analysis
RAPD is used for the genetic variability and phylogenetic studies of
diverse organisms for resolving taxonomic problems. RAPD detects
polymorphisms with a sole primer of arbitrary nucleotide sequence.
RAPD has the benefit that no previous information of the genome
is needed for effective application. RAPD approach is a comparatively straightforward technique to apply, and the number of loci
that can be scrutinized is unlimited.
5.10.1 Materials
Required
l
Source: Nematode DNA.
l
Equipment: Spectrophotometer, pH meter, vortex, refrigerator,
electrophoresis unit, PCR, gel documentation.
l
Chemicals: PCR mixture.
l
PCR mixture consists of:
– Taq DNA polymerase: 1.5 U.
– dNTPs (2.5 mM each): 2 μl.
– Taq buffer (10Â): 2.5 μl.
– MgCl 2 : 2 Mm.
– Primer: 0.5 μM.
– Genomic DNA: 100 ng.
– Double distilled to make up the volume 25 μl.
5.10.2 Procedure
1. The nucleotide sequences of RAPD primers are shown in the
Table 1.
2. Carry out the PCR in 25 μl volume containing PCR mixture.
3. Preheat the reaction mixture at 95
C and incubate for 5 min at
94
C.
Molecular Characterization
91