12. Switch on UV light.
13. Photograph the DNA seen with an orange color bands.
4.5 PCR
Amplification
1. Amplify the separated DNA segment containing the 16S rDNA
gene (1.5 kb) by polymerase chain reaction (PCR) using primers 27F (5
0 -AGA GTT TGA TCC TGG CTC AG-3
0 ) and
1492R (5
0 -GGT TAC CTT GTT ACG ACT T-3
0 ).
2. The PCR mixture (50 μl) consists of 10 mM Tris–HCl
(pH 8.3), 50 mM KCl, 2 mM MgCl 2 , 0.2 mM dNTPs,
0.2 pmol of each primer, and Taq DNA polymerase.
3. Perform amplification step by practicing a protocol consisting
of 30 cycles that included 1 min of denaturation at 95
C, 30 s
of annealing at 55
C, 1 min of elongation at 72
C, and a final
extension of 5 min at 72
C.
4. Run the amplified product on agarose gel electrophoresis.
5. Purify the PCR product (PCR purification kit).
6. Conduct sequencing by a DNA sequencing machine.
7. Edit the 16S rDNA gene sequences obtained from the isolated
bacterial strain using a multiple sequence editor.
8. Investigate the nucleotide comparison using the BLASTN program from the National Center for Biotechnology Information
(NCBI).
9. Align the nucleotide sequences using ClustalW2 from EBI
(http://www.ebi.ac.uk/Tools/clustalw2).
10. Compute pair-wise evolutionary distances using Poisson correction for multiple substitutions.
11. Construct the phylogenetic tree of the isolated strain using the
maximum parsimony method in the Mega software program.
5 Evaluation of Bioactive Compound of Symbiotic Bacteria
5.1 Extraction of
Bioactive Compound
1. Transfer a single colony of Xenorhabdus and Photorhabdus on
NBTA. Later, culture it in a 1000-ml flask containing
500 ml LB.
2. Incubate the inoculated culture at room temperature for 72 h
on a rotary shaker at 28
C.
3. Add 1000 ml of ethyl acetate to the inoculated culture and
blend well.
4. Allow the flask to stay at room temperature for 24 h.
5. Concentrate the bacterial extracts using a rotary vacuum
evaporator.
Evaluation of Bioactive Compound of Symbiotic Bacteria
137
13. Photograph the DNA seen with an orange color bands.
4.5 PCR
Amplification
1. Amplify the separated DNA segment containing the 16S rDNA
gene (1.5 kb) by polymerase chain reaction (PCR) using primers 27F (5
0 -AGA GTT TGA TCC TGG CTC AG-3
0 ) and
1492R (5
0 -GGT TAC CTT GTT ACG ACT T-3
0 ).
2. The PCR mixture (50 μl) consists of 10 mM Tris–HCl
(pH 8.3), 50 mM KCl, 2 mM MgCl 2 , 0.2 mM dNTPs,
0.2 pmol of each primer, and Taq DNA polymerase.
3. Perform amplification step by practicing a protocol consisting
of 30 cycles that included 1 min of denaturation at 95
C, 30 s
of annealing at 55
C, 1 min of elongation at 72
C, and a final
extension of 5 min at 72
C.
4. Run the amplified product on agarose gel electrophoresis.
5. Purify the PCR product (PCR purification kit).
6. Conduct sequencing by a DNA sequencing machine.
7. Edit the 16S rDNA gene sequences obtained from the isolated
bacterial strain using a multiple sequence editor.
8. Investigate the nucleotide comparison using the BLASTN program from the National Center for Biotechnology Information
(NCBI).
9. Align the nucleotide sequences using ClustalW2 from EBI
(http://www.ebi.ac.uk/Tools/clustalw2).
10. Compute pair-wise evolutionary distances using Poisson correction for multiple substitutions.
11. Construct the phylogenetic tree of the isolated strain using the
maximum parsimony method in the Mega software program.
5 Evaluation of Bioactive Compound of Symbiotic Bacteria
5.1 Extraction of
Bioactive Compound
1. Transfer a single colony of Xenorhabdus and Photorhabdus on
NBTA. Later, culture it in a 1000-ml flask containing
500 ml LB.
2. Incubate the inoculated culture at room temperature for 72 h
on a rotary shaker at 28
C.
3. Add 1000 ml of ethyl acetate to the inoculated culture and
blend well.
4. Allow the flask to stay at room temperature for 24 h.
5. Concentrate the bacterial extracts using a rotary vacuum
evaporator.
Evaluation of Bioactive Compound of Symbiotic Bacteria
137