4.1.3 Preparation of
Symbiotic Bacteria Culture
1. Take 2.5 ml nutrient broth in 100-ml conical flask.
2. Add 1 ml of broth culture or single colonies and mix well.
3. Incubate at 37
C for 24 h.
4.1.4 Procedure
1. Take 2 ml overnight culture.
2. Harvest the cells by centrifugation at 15,000 g for 10 min.
3. Add 875 μl of TE buffer to the cell pellet.
4. Re-suspend the cells in the buffer by gentle mixing.
5. Add 100 μl of 10% SDS and 5 μl of proteinase K to the cells.
6. Mix the above combination well.
7. Incubate the mixture at a temperature of 37ºC for an hour in
an incubator.
8. Add 1 ml of phenol–chloroform mixture to the substances and
blend well by inverting.
9. Incubate the mixture at room temperature for 5 min.
10. Centrifuge the mixture at 15,000 g for 10 min at 4ºC.
11. Collect the extremely viscous jelly like supernatant using cut
tips and allocate them to a fresh tube.
12. Repeat the procedure once again with phenol–chloroform
mixture.
13. Collect the supernatant gently in a fresh tube.
14. Add 100 μl of 5 M sodium acetate to the supernatant and blend
gently.
15. Add 2 ml of isopropanol and mix gradually by inversion till
white strands of DNA precipitates out.
16. Centrifuge the mixture at 3000 x g for 10 min.
17. Eliminate the supernatant and add 1 ml 70% ethanol.
18. Centrifuge the above mixture at 3000 x g for 10 min.
19. Add 200 μl of TE buffer after air drying the contents for 5 min
and store at 4
C.
20. Take 10 μl of DNA sample dilute to 1 or 2 ml with distilled
water.
21. Determine the DNA concentration using a spectrophotometer
at 260/280 nm.
22. Store DNA at À20
C for further use.
4.2 Quantification
of DNA
This experiment is virtuously an application of the Beer Lamberts’
Law which states that “the concentration of the sample is directly
proportional to the absorbance of light done by the sample. It is
given by following expression: A ¼ € * C * l.” The device UV
spectrophotometer works on the above principle and employed to
134
Laboratory Techniques for Symbiotic Bacteria
Symbiotic Bacteria Culture
1. Take 2.5 ml nutrient broth in 100-ml conical flask.
2. Add 1 ml of broth culture or single colonies and mix well.
3. Incubate at 37
C for 24 h.
4.1.4 Procedure
1. Take 2 ml overnight culture.
2. Harvest the cells by centrifugation at 15,000 g for 10 min.
3. Add 875 μl of TE buffer to the cell pellet.
4. Re-suspend the cells in the buffer by gentle mixing.
5. Add 100 μl of 10% SDS and 5 μl of proteinase K to the cells.
6. Mix the above combination well.
7. Incubate the mixture at a temperature of 37ºC for an hour in
an incubator.
8. Add 1 ml of phenol–chloroform mixture to the substances and
blend well by inverting.
9. Incubate the mixture at room temperature for 5 min.
10. Centrifuge the mixture at 15,000 g for 10 min at 4ºC.
11. Collect the extremely viscous jelly like supernatant using cut
tips and allocate them to a fresh tube.
12. Repeat the procedure once again with phenol–chloroform
mixture.
13. Collect the supernatant gently in a fresh tube.
14. Add 100 μl of 5 M sodium acetate to the supernatant and blend
gently.
15. Add 2 ml of isopropanol and mix gradually by inversion till
white strands of DNA precipitates out.
16. Centrifuge the mixture at 3000 x g for 10 min.
17. Eliminate the supernatant and add 1 ml 70% ethanol.
18. Centrifuge the above mixture at 3000 x g for 10 min.
19. Add 200 μl of TE buffer after air drying the contents for 5 min
and store at 4
C.
20. Take 10 μl of DNA sample dilute to 1 or 2 ml with distilled
water.
21. Determine the DNA concentration using a spectrophotometer
at 260/280 nm.
22. Store DNA at À20
C for further use.
4.2 Quantification
of DNA
This experiment is virtuously an application of the Beer Lamberts’
Law which states that “the concentration of the sample is directly
proportional to the absorbance of light done by the sample. It is
given by following expression: A ¼ € * C * l.” The device UV
spectrophotometer works on the above principle and employed to
134
Laboratory Techniques for Symbiotic Bacteria