Process Flow of PCR Analysis with DNA Test Kits – Step by Step Procedure

Extraction

  • Obtain SAMPLE [For Animal (Shrimp) sample, use less than 10mg.] —> HOMOGENIZE manually.
  • Add 40ul of the GenCheck extraction Reagent.
  • Vortex.
  • Centrifuge at 10000 rpm for 30 seconds.
  • Heat block (Thermo-Shaker) at 98-100 °C for 10 minutes.
  • Stay on ice for 1 minute.
  • Centrifuge at 10000 rpm for 5 minutes.
  • Transfer 5 ul of the Supernatant liquid to a new 1.5ml tube.
  • Add 95ul of the RNase-free water.

Amplification

For Sample

  • Prepare the PCR mix in a 1.5ml tube as follows:

         PCR enzyme     5ul X
     RNase-free water    2ul X
     Primer solution     2ul X

  • Vortex the PCR mix.
  • Put 9ul of the PCR mix into each PCR tube.
  • Put 1ul of the sample DNA or 1ul of the Positive Control into each PCR tube.
  • Multi spin.
  • Perform the PCR step.

VERY IMPORTANT Notes:

  • The PCR Cooler Set, the PCR enzyme and the Positive Controls MUST be FROZEN (stored at – 20°C).
  • All Reagents for example, the RNase-free water and the Primer solutions, MUST be REFRIGERATED (stored at +2°C to +8°C) or in COLD temperature
  • Put the 1.5 or 2.0 ml Tube Rack on TOP of an ICE PACK or on TOP of ICE during use or when preparing the PCR reagants and the PCR mix.

Identification

IDENTIFICATION of the Shrimp Pathogen(s):

  • Prepare the Latex Mixture below (in a 1.5ml tube) as follows:

         Latex solution     1.5ul X
     Dilution Buffer     20ul X

  • Centrifuge the above Latex Mixture.
  • Put 21.5 ul of the Latex Mixture into a tube after the PCR step.
  • After this, put a C-PAS Strip into each PCR tube.
  • Wait for 10 minutes.
  • And finally, visually IDENTIFY the SHRIMP PATHOGEN(S) that appear as BLUE line(s) on the C-PAS Strip or by placing the C-PAS Strip(s) into the C-PAS Card or Image READER.