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The steps listed below need to be followed when setting up for the hybridisation procedure.

 

Workstation Set-up

  • Compile all the tools and reagents required for the step.
  • Switch on all the equipment and check whether they are in working condition.
  • Always connect the Twincubator and GT blot to the UPS.
  • Thoroughly clean the workbench and tools with appropriate disinfectant.
  • Clean the equipment as suggested by the manufacturer.
  • Place an absorbent sheet.
  • Bring out the hybridization reagents from the refrigerator.

Reagent Preparation

  • Kit Component 1 is used for this step.​
  • Hybridization reagent preparation and steps involved in hybridization procedure remains the same in First Line (FL) and Second Line (SL) Line Probe Assay (LPA).
  • Bring out the reagent kit from the refrigerator.
  • Pre-warm the hybridisation and stringent solution in a water bath, as these 2 solutions form crystals due to the kit’s storage at 2 - 8ºC.

 

Reagents Required for Hybridization 

  1. Hybridization Solution - HYB​
  2. Stringent Solution - STR​
  3. Conjugate Solution - CON​
  4. Substrate Solution - SUB​
  5. RIN Solution - RIN​
  6. Distilled Water - DW​

 

The steps for preparing these reagents are shown in the tables below.

 

Table 1: Preparation of Hybridization and Stringent Solutions
PREPARATION OF HYBRIDIZATION SOLUTION PREPARATION OF STRINGENT SOLUTION
  • Pre-warm hybridization solution in a water bath.
  • Check the hybridization solution for precipitate.
  • If the solution is consistent, without any precipitate, aliquot the calculated amount to a falcon tube and label it as “HYB".
  • Remember: Hybridization solution should be warm when adding it to the Hybridization tray.
  • Pre-warm stringent solution in a water bath.
  • Check the stringent bottle for precipitate.
  • If the solution is consistent, without any precipitate, aliquot the calculated amount to a falcon tube and label it as “STR”. 

 

Table 2: Preparation of Conjugate and Substrate Solutions
PREPARATION OF CONJUGATE SOLUTION PREPARATION OF SUBSTRATE SOLUTION
  • Aliquot calculated conjugate diluent to a 50 ml falcon using a micropipette.
  • Add calculated conjugate concentrate to it, using a micropipette.
  • Mix well.
  • Label it as “CONJUGATE”.
  • Aliquot calculated substrate diluent to a 50 ml falcon using a micropipette.
  • Add calculated substrate concentrate to it, using a micropipette.
  • Mix well.
  • Cover the falcon tube with aluminium foil as the substrate is photosensitive.

 

Table 3: Preparation of RIN Solution and Distilled Water
PREPARATION OF RIN SOLUTION FOR DISTILLED WATER
  • Aliquot calculated RIN solution to a 50 ml falcon tube.
  • Label it as "RIN".
  • Aliquot the calculated amount of distilled water required in a falcon tube.
  • Label it as "DW".

 

Please click the video below to see more on hybridization.

 

https://empowerschoolofhealth-my.sharepoint.com/:v:/g/personal/admin_empowerschoolofhealth_org/EfnhWStUA7tJu8XdrVc9AXABiFl_5EO8LK-x_jx7dqxBzg?e=bGXgAr

 

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