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General instructions when conducting hybridization using a twincubator are:

 

  • Thoroughly clean the workbench with disinfectant​ (1% sodium hypochlorite).
  • Prepare reagents as per the calculation table.
  • Using clean/ new tweezers place strips onto a clean sheet of paper and label them as in the worksheet.​
  • Use a clean/ new tray.

 

Steps of Hybridization Using Twincubator​ 

Step-1
  • Dispense 20 μl denaturation (DEN) solution in the corner of each trough.

Note: No need to change tips between samples.

Step-2
  • Add 20 µl Amplicon to DEN and carefully mix by pipetting up and down.​
  • Incubate for 5 min​utes.

Note: Change tips between samples.​

Step-3
  • Carefully add 1 ml of pre-warmed hybridization (HYB) buffer to each well, using filtered tips.

Note: Change tips between samples.

  • Gently shake the tray to homogenously mix HYB with amplicon.​
  • Do not spill into neighbouring troughs.​
Step-4
  • Carefully place numbered strips in each well, strip face side up using clean/ new plastic tweezers.​
  • Strips must be completely immersed in HYB.
  • If strips turn over, reposition them with a fresh pipette tip.
Step-5
  • Place tray onto Twincubator and press START.
  • Twincubator will then start shaking with a temperature of 45°C.​
  • This step will be of 30 minutes.
  • After 30 minutes, the Twincubator will set off an alarm.
  • When the alarm goes off, press the right arrow to stop.
Step-6
  • Completely aspirate HYB using transfer pipettes.

Note: 

​Change pipettes for every sample.

Do not decant the contents of the tray into a sink or jar as this can contaminate the work area.

Wipe off the condensation that forms on the lid of the incubator before every incubation step.

Step-7
  • Add 1 ml of STR solution to each well.
  • Press start.
  • This step will be of 15 minutes at 45°C.​
  • After 15 minutes, the Twincubator will set off an alarm.
  • When the alarm goes off, press the right arrow to stop​
Step-8
  • Completely aspirate STR solution by transfer pipette.

Note: ​

Change pipettes for every sample.

Do not decant the contents of the tray into a sink or jar; this can contaminate the work area.

Step-9
  • Add 1ml of RIN solution to each well.​
  • Press start.
  • This step will be for 1 minute.
  • After 1 minute, the machine will set off an alarm.
  • Once the alarm goes off, press the right arrow to stop.
Step-10
  • Completely aspirate the RIN solution by transfer pipette.​

Note: ​No need to change pipette between wells.

  • From this step, the content of the tray may be decanted into a container with 1% sodium hypochlorite solution.
Step-11
  • Add 1 ml of conjugate solution to each well using a micropipette.

Note: No need to change tips for every sample.​

  • Press start.
  • This step will be of 30 minutes.
  • After 30 minutes, the machine will set off an alarm.​ 
  • Once the alarm goes off, press the right arrow to stop.
Step-12
  • Completely aspirate conjugate solution using a transfer pipette, or the content of the tray may be decanted into a container with 1% freshly prepared sodium hypochlorite solution.
Step-13
  • Work at room temperature from this step.
  • Add 1 ml of RIN solution to each well.
  • Press start.
  • This step will be for 1 minute.
  • After 1 minute, the machine will set off an alarm.
  • Once the alarm goes off, press the right arrow to stop.
Step-14
  • Completely aspirate the RIN solution using a transfer pipette, or the content of the tray may be decanted into a container with 1% freshly prepared sodium hypochlorite solution.
Step-15
  • Repeat step-13.
Step-16
  • Repeat step-14
Step-17
  • Add 1 ml of distilled water to each well.
  • Press start.
  • This step will be for 1 minute.
  • After 1 minute, the machine will set off an alarm.
  • Once the alarm goes off, press the right arrow to stop.
Step-18
  • Completely aspirate distilled water using a transfer pipette, or the content of the tray may be decanted into a container with 1% freshly prepared sodium hypochlorite solution.
Step-19
  • Add 1 ml of substrate solution to each well.

Note: No need to change tip.

  • Cover the machine with aluminium foil as the substrate solution is photosensitive.
  • Press start.
  • This step may be for 3-5 minutes (depending on the colour that develops). ​
  • The machine will set off an alarm.
  • Once the alarm goes off, press the right arrow to stop.
Step-20
  • Completely aspirate the substrate solution using a transfer pipette, or the content of the tray may be decanted into a container with 1% freshly prepared sodium hypochlorite solution.
Step-21
  • Add 1 ml of distilled water to each well.
  • Press start.
  • This step will be for 1 minute.
  • After 1 minute, the machine will set off an alarm.
  • Once the alarm goes off, press the right arrow to stop.
Step-22
  • Completely aspirate distilled water using a transfer pipette, or the content of the tray may be decanted into a container with 1% freshly prepared sodium hypochlorite solution.
Step-23
  • Repeat step-21.
Step-24
  • Do not aspirate distilled water from wells.
Step-25
  • Using clean tweezers, remove the strips from the tray and dry them between two layers of absorbent paper​
Step-26
  • Align strips and paste them on a reporting sheet using cello tape/ scotch tape.

 

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