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Mycoviral double-stranded RNA extraction from plant leaf with Minilys

Instrument: Minilys homogenizer

Sample:  Green pepper (Capsicum annum) leaf and Malabar spinach (Basella alba) leaf

Protocol:

  • Precellys kit: 03961-1-002 (2.8 mm ceramic beads).
  • Add Samples: 20 mg of green pepper (Capsicum annum) leaf and 20 mg of Malabar spinach (Basella alba) leaf.
  • Add Buffer: 500μL of STE buffer 2X.
  • Run the Minilys at 5000 rpm, 60 sec x 1, 2 or 3 cycles with no time break on ice between each cycles.
  • Addition of SDS ; phenol-chloroform extraction.

Results:

A protocol of 5000 rpm x 60 sec with CK28 gave the best results for both green pepper and Malabar spinach leaves homogenization. Extension of the time homogenization attenuated the dsRNA band of mycoviral especially in the Malabar spinach sample.

 
Agarose gel electrophoresis of 5 mg extracted plant