Hybridization Using GT BLOT in LPA Lab Settings

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General Instructions

Thoroughly clean the work bench with 1% sodium hypochlorite solution.
Run the wash cycle A and B in GT Blot.
After cleaning, prepare reagents as per the calculation table.
Pour prepared reagents into reagent bottles provided with GT Blot.
Place GT Blot reagent bottles back to machine.
Cover the bottles with a tubed lid.
Using clean/ new tweezers, place strips onto a clean sheet of paper and label them same as that in the work sheet.
Use a clean/ new tray, check for any wear/ tear.

Steps for hybridization using twincubator are shown in the table below.

Table: Hybridization Steps using Twincubator
STEP-1	DISPENSE 20ΜL DENATURATION (DEN) SOLUTION IN A CORNER OF EACH WELL NOTE: NO NEED TO CHANGE TIPS
Step-2	Add 20µl amplicon to DEN and carefully mix by pipetting up and down Incubate for 5 min Change tips for every sample Carefully place the tray in GT Blot Close the GT Blot lid Press start Select no. of wells (always in even numbers) Press start Note: Change tips between samples
Step-3	The instrument will automatically dispense hybridization solution to each well selected Message – ‘Add amplicon’ will be displayed
Step-4	Carefully place numbered strips in each well, up (face-side), using clean/ new plastic tweezers Close the lid of GT Blot Press start All the subsequent steps will be carried out automatically in a fully automated GT Blot machine

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