Hybridization Using Twincubator in LPA Lab Settings

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.