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Protocols |
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COOKE
AND DUNCAN (1997)PROTOCOL FOR GENOMIC DNA ISOLATION PHYTOPHTHORA |
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- Four days old young mycelium grown in Reibeiro's liquid
medium was filtered through sterile filter paper.
- Dried and weighed
- 0.1g of mycelium was taken in an eppendorf tube with 50mg
of sterile sand or glass powder and 10mg PVPP
- 750µl extraction buffer was added to the eppendorf tube
.
- The mixture was ground.
- Centrifuged at 13,000 rpm for 5 minutes.
- After centrifugation the supernatant was taken in a sterile
eppendorf.
- 500µl Tris phenol: chloroform: isoamyl alcohol (25:24:1)
was added to it and inverted gently for 2 minutes.
- Centrifuged at 13,000 rpm for 5 minutes.
- The aqueous layer was transferred to sterile eppendorf
tube.
- Fill the tube with isopropanol and gently inverted.
- Centrifuged at 13,000rpm for 10 minutes.
- Centrifuged at 13,000 rpm for 2 minutes.
- Air dried the pellet.
- Resuspended I 100µl sterile distilled water.
- 3µl RNase (5mg/ml) added and incubated for 30 minutes
at 370C.
- Stored at -200C.
- Agarose Gel electrophoresis carried out with 0.8% agarose
gel, added 8µl ethidium bromide (10µg/ml) into 200ml 1X
TAE buffer.
- DNA quantified by using eppendorf biophotometer.
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| Stock Chemicals for DNA Isolation |
- 1 M Tris HCl
Tris base : 12.1g
Distilled water : 100 ml
pH : 7.5
- 5M NaCl
NaCl : 29.22g
Distilled water : 100 ml
- 0.5M EDTA
EDTA : 18.61g
Distilled water : 100 ml
pH : 8.0
- 10% SDS
SDS :10 g
Distilled water : 100 ml
pH : 7.2
- STE Extraction Buffer (50ml)
1 M Tris HCl : 10 ml
5M NaCl : 2.5 ml
0.5M EDTA : 2.5 ml
10% SDS : 2.5 ml
Distilled water : 32.5 ml
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