Untitled Document

P h y t o p h t h o r a ....D i s e a s e s.... i n .....H o r t i c u l t u r a l ....C r o p s
 
   
   Phyto finder
 Species
 Crops
 Cultures
 Genome Resources
 Primer Database
 Genome Database
 Protocols
 Structure
 Submit
Untitled Document
Protocols
  
   Protocols (Phytophthora Related)
  DNA isolation from Black Pepper (2% CTAB, modified Doyle and Doyle protocol)
  Genomic DNA isolation of Phytophthora(Cooke and Duncan (1997) protocol)
  Detection and isolation of phytophthora from soil(ANANDARAJ & SARMA, 1990)
  Protocol for isolation of phytophthora (Erwin & Ribeiro, 1996)
  Protocol for determination of mating type of phytophthora (Erwin & Ribeiro, 1996)
  Protocol for isolation of endophytic fungi from black pepper((Arnold et al 2001)
    Protocols (Ralstonia Related)
  Detection Method for Ralstonia Solancearum in Soil
  Culturing Methods-Ralstonia Solancearum
   Protocols (Nematodes Related)
  Nematode Extraction
  Culturing of Nematodes
ISOLATION OF DNA FROM BLACK PEPPER (2% CTAB, MODIFIED DOYLE AND DOYLE PROTOCOL)         Download
  • Pre-heated 10 ml extraction buffer with 50µl 0.5% Beta-mercaptoethanol to 600C.
  • Ground 2g tissue to fine powder with liquid nitrogen. Added 50 mg PVPP and mixed. Transferred the contents into the centrifuge tube containing 10ml CTAB buffer pre-heated to 600C and shaken gently.
  • Incubated the tube for 1 hr at 600C, shaken intermittently in every 10 minutes and cooled to room temperature.
  • Added 10ml of chloroform-isoamyl alcohol (24:1) and mixed gently by inverting tubes about 25 times to form an emulsion.
  • Spun at 5000rpm for 15 minutes and transferred aqueous phase to new centrifuge tube using cut tips. If cloudy added 6ml of chloroform-isoamyl alcohol and repeated the step.
  • Transferred clear aqueous phase to fresh centrifuge. Added 2.5 ml 5M NaCl and mixed.
  • Added 10ml cold ethanol, mixed gently and refrigerated overnight at -200C.
  • Centrifuged at 3000rpm for 3 minutes,then increased the speed to 5000rpm for 3 minutes at room temperature. Poured off the supernatant, washed the pellet with 2ml 76% ethanol, centrifuged as above for 3 minutes.
  • Repeated washing twice
  • Drained out the supernatant. Removed ethanol without completely drying DNA by leaving tubes uncovered at 370C for 20-30 minutes or vacuum dry at room temperature.
  • Re-suspended the pellet in 1ml TE and pppoled by using cut tips.
  • Added RNAase to a final concentration of 10µg/ml. Incubated at 370C for 30 minutes.
  • 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.
Under the authority of IISR, developed by the Bioinformatics Centre