extension, to confirm that all of the final amplification products
are full length.
6. Store the amplified samples at 4
C.
5.5 Agarose Gel
Electrophoresis
1. Mix PCR amplified product (10 μl) with 2 μl loading buffer
(0.1% bromophenol blue, 0.25% xylene cyanol, 40% w/v
sucrose in water).
2. Run on 2.5% agarose gel, containing 1 μg/ml ethidiumbromide, at room temperature and 60 V for 3 h in TBE
running buffer.
3. Use a 100 bp DNA marker as reference.
4. View the gel under gel documentation.
5.6 Sequencing
1. Purify the PCR products with a PCR purification kit.
2. Sequence the purified DNA in both directions on automated
sequencers.
3. Use DNA sequencer for sequence editing and authenticating
base calls.
5.7 RFLP Analysis
Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) of the ribosomal DNA (rDNA) repeat unit is
extensively used for the documentation of entomopathogenic
nematodes. It helps in detection within total genomic DNA. It is
a supreme preference for identification purposes, since it is present
as a multi-copy tandem repeat in the genome of many organisms.
5.8 Restriction
Digestion and
Electrophoresis of PCR
Products [175]
To identify the nematode isolates by PCR-RFLP of the ITS region
(internal transcribed spacer) of the ribosomal DNA.
5.8.1 Materials Required
Source: Nematode DNA.
Equipment: PCR machine, electrophoresis unit, UV light
lamp, micropipette.
Chemicals/reagents: Digestion enzymes, agarose, TBE buffer,
ethidium bromide.
5.8.2 Procedure
1. Digest 5 μl amplified ITS PCR products with 0.5 μl selected
restriction endonucleases (e.g., Pst I; Pvu II; Sau 3 AI; Sal I;
Alu I; Hpa II; Hha I; Hind III; Hinf I; Xha I).
2. Carry all digestions at 37
C for 3 h.
3. Separate the products of the restriction digestion by electrophoresis in 2% agarose gels (w/v), in a 1Â TBE buffer at 100 V
for 2 h.
90
Laboratory Techniques for Entomopathogenic Nematodes
are full length.
6. Store the amplified samples at 4
C.
5.5 Agarose Gel
Electrophoresis
1. Mix PCR amplified product (10 μl) with 2 μl loading buffer
(0.1% bromophenol blue, 0.25% xylene cyanol, 40% w/v
sucrose in water).
2. Run on 2.5% agarose gel, containing 1 μg/ml ethidiumbromide, at room temperature and 60 V for 3 h in TBE
running buffer.
3. Use a 100 bp DNA marker as reference.
4. View the gel under gel documentation.
5.6 Sequencing
1. Purify the PCR products with a PCR purification kit.
2. Sequence the purified DNA in both directions on automated
sequencers.
3. Use DNA sequencer for sequence editing and authenticating
base calls.
5.7 RFLP Analysis
Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) of the ribosomal DNA (rDNA) repeat unit is
extensively used for the documentation of entomopathogenic
nematodes. It helps in detection within total genomic DNA. It is
a supreme preference for identification purposes, since it is present
as a multi-copy tandem repeat in the genome of many organisms.
5.8 Restriction
Digestion and
Electrophoresis of PCR
Products [175]
To identify the nematode isolates by PCR-RFLP of the ITS region
(internal transcribed spacer) of the ribosomal DNA.
5.8.1 Materials Required
Source: Nematode DNA.
Equipment: PCR machine, electrophoresis unit, UV light
lamp, micropipette.
Chemicals/reagents: Digestion enzymes, agarose, TBE buffer,
ethidium bromide.
5.8.2 Procedure
1. Digest 5 μl amplified ITS PCR products with 0.5 μl selected
restriction endonucleases (e.g., Pst I; Pvu II; Sau 3 AI; Sal I;
Alu I; Hpa II; Hha I; Hind III; Hinf I; Xha I).
2. Carry all digestions at 37
C for 3 h.
3. Separate the products of the restriction digestion by electrophoresis in 2% agarose gels (w/v), in a 1Â TBE buffer at 100 V
for 2 h.
90
Laboratory Techniques for Entomopathogenic Nematodes