TBE buffer
Tris borate (1Â TBE)
10Â buffer/lit
89 mm Tris base
108.0 g
89 mm Boric acid
55.0 g
25 mm Na 2 -EDTA
9.3 g
TAE buffer
Tris borate (1Â TBE)
10Â buffer/lit
50 mm Tris base
302.5 g
25 mm acetic acid
71.4 g
1 mm Na 2 -EDTA
18.6 g
Sterilize the stock solutions by autoclaving.
l
Preparation of ethidium bromide (stock solution).
Weigh 10 mg ethidium bromide. Transfer it into a sterile
tube and dissolve in 10 ml sterile distilled water. The stock is
stored at 4
C.
l
Preparation of sample loading dye.
Mix glycerol and bromophenol blue (6Â), 3 ml glycerol
(30%), 25 mg bromophenol blue (0.25%) in distilled water to
10 ml.
4.4.2 Procedure
Preparation of Agarose Gel
1. Dissolve the agarose (0.5 g) in 50 ml of 1Â TAE. This gives 1%
agarose gel.
2. Boil agarose to dissolve completely to a clear solution.
3. Cool agarose to lukewarm (60
C) and add 0.5 μg/ml ethidium bromide.
4. Place the comb of electrophoresis set, approximately 2 cm away
from the cathode.
5. Dispense the agarose without making any bubbles, cool it for
20 min. The thickness of the gel should be around 0.5–0.9 cm.
6. Add 1Â TAE buffer into the gel tank till the buffer level stands
at 0.5–0.8 cm above the gel surface and gently take off
the comb.
7. Connect the power cord to the electrophoretic power pack
setup and run the gel for 1 h at 100 V.
8. Blend the DNA sample (100–200 ng) with the loading dye
(5 μl DNA + 1 μl dye) and load into the well using a pipetman.
9. Switch off the current when the bromophenol blue (the tracking dye) has moved 1 cm above the bottom end.
10. Disconnect the power supply after about 30–45 min, 50 V.
11. The gel is gently pushed onto the UV transilluminator.
136
Laboratory Techniques for Symbiotic Bacteria
Tris borate (1Â TBE)
10Â buffer/lit
89 mm Tris base
108.0 g
89 mm Boric acid
55.0 g
25 mm Na 2 -EDTA
9.3 g
TAE buffer
Tris borate (1Â TBE)
10Â buffer/lit
50 mm Tris base
302.5 g
25 mm acetic acid
71.4 g
1 mm Na 2 -EDTA
18.6 g
Sterilize the stock solutions by autoclaving.
l
Preparation of ethidium bromide (stock solution).
Weigh 10 mg ethidium bromide. Transfer it into a sterile
tube and dissolve in 10 ml sterile distilled water. The stock is
stored at 4
C.
l
Preparation of sample loading dye.
Mix glycerol and bromophenol blue (6Â), 3 ml glycerol
(30%), 25 mg bromophenol blue (0.25%) in distilled water to
10 ml.
4.4.2 Procedure
Preparation of Agarose Gel
1. Dissolve the agarose (0.5 g) in 50 ml of 1Â TAE. This gives 1%
agarose gel.
2. Boil agarose to dissolve completely to a clear solution.
3. Cool agarose to lukewarm (60
C) and add 0.5 μg/ml ethidium bromide.
4. Place the comb of electrophoresis set, approximately 2 cm away
from the cathode.
5. Dispense the agarose without making any bubbles, cool it for
20 min. The thickness of the gel should be around 0.5–0.9 cm.
6. Add 1Â TAE buffer into the gel tank till the buffer level stands
at 0.5–0.8 cm above the gel surface and gently take off
the comb.
7. Connect the power cord to the electrophoretic power pack
setup and run the gel for 1 h at 100 V.
8. Blend the DNA sample (100–200 ng) with the loading dye
(5 μl DNA + 1 μl dye) and load into the well using a pipetman.
9. Switch off the current when the bromophenol blue (the tracking dye) has moved 1 cm above the bottom end.
10. Disconnect the power supply after about 30–45 min, 50 V.
11. The gel is gently pushed onto the UV transilluminator.
136
Laboratory Techniques for Symbiotic Bacteria