Bionalyzer pico kit
Material :
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Rnase-free water
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Pipettes
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0,5 ml tubes Rnase free
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Heating block
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Pico kit and reagents
Primer preparation (Only before the first run)
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Spin ladder down.
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Heat denature the ladder for 2 min at 70 °C
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Immediately cool down the vial on ice
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Add 90 uL of Rnase-free water and mix thoroughly
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Prepare aliquots of ladder
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Store aliquots at -70°C (see « Pico ladder » tubes at -70°C)
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Before use, thaw ladder aliquots and keep them on ice.
Chip Priming station
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Take the priming station on the Bioanalyzer room
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Inser your syringe from the clip.
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Slide it into the hole of the luer lock adapter and screw it tightly to the chip priming station.
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Open the chip priming station by pulling the latch
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Using a screwdriver, open the screw at the underside of the base plate.
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Lift the base plate and insert it again in position C. Retighten the screw.
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Now, adjust the syringe clip : release the lever of clip and slide it up to the top position.
Bioanalyzer preparation (use the transparent RNA chIP to clean the analyzer)
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Fill one of the wells of the chip cleaner with 350 uL of fresh Rnase-free water
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Turn on the computer into the bioanalyzer room : agilent session ; password : banalyze.
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Open the program and open the lid of the analyzer.place the electrode cleaner in the Agilent 2100 Bioanalyzer
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Close the lid and leave it closed for 5 minutes (or more. During this time , prepare your computer assay and set the name of the samples on computer).
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Open the lid and remove the electrode cleaner ( ! there is only one cleaner chip so keep it for future use).
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Wait 30 sec to allow the water on the elecrodes to evaporate before closing the lid.
Preparing the Gel (before this step look at 4°C if aliquots are ready: if this is the case, proceed to next step )
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Allow all reagents to equilibrate to room temperature for 30 minutes before use.
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Place 550 ul of RNA 6000 pico gel matrix (red cap) into the top receptacle of a spin filter.
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Spin the filter tube for 10 min at 4000 rpm
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Aliquot 65 uL fileterd gel into 0,5 mL Rnase-free tubes that are included in the kit (you normaly obtain 7 aliquots). Store the aliquots at 4°C and use them within one month of preparation (see « pico gel » tubes at 4 °C.)
Preparing the Gel-Dye Mix
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Allow all reagents to equilibrate to room temperature for 30 minutes befor use. Protect the dye concentrate from light.
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Vortex RNA 6000 Pico dye concentrate (blue cap) for 10 sec and spin down.
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Add 1 uL of RNA 6000 Pico dye concentrate (blue cap) to 65 uL aliquot of filtered gel.
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Vortex thoroughly and store the dye concentrate at 4 °C in the dark again.
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Spin tube for 10 minutes at room temperature (14 000 rpm : During this time prepare your sample dilutions ). Use prepared gel-dye mix within one day.( each tube allows for two chips)
Loading the Gel-Dye mix
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Take a new RNA pico chip out of its sealed bag.
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Place the chip on the chip priming station.
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Pipette 9 uL of the gel-dye mix at the bottom of the well marked G and dispense the gel dye mix with the next steps.
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Set the timer to 30 seconds, make sure that the plunger is positioned at 1 ml and then close the chip
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priming station. The lock of the latch will click when the chip priming station is closed correctly.
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Press the plunger of te syrunge down until it is held by the clip.
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Wait for exactly 30 sec and release the plunger moves back at least to the 0,3 mL mark.
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Wait for 5 sec then slowly pull back the plunger to the 1 ml position.
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Open the chip priming station.
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Pipette 9 uL of the gel dye mix in each of the wells marked G
Loading solution and marker
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Pipette 9 uL of the RNA 6000 pico conditioning solution (white cap) into the well marked CS
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Pipette 5 uL of the RNA 6000 pico marker (green cap) into the well marked with a ladder symbol (DNA molecule symbol) and each of the 11 sample wells.
!!! Do not leave any wells empty : Add 5 ul of the RNA marker (green cap)plus 1 uL of water to each unused sample well.
Loading ladder and samples.
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To minimize secondary structure, you may heat denature 70°C for 2 min the samples before loading them on the chip.
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Pipette 1 ul of the diluted RNA 6000 pico Ladder into the well marked with the ladder sybol (DNA molecule symbol) .
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Pipette 1 uL of each sample into each of the 11 sample wells (RNA concentration accepted: 50-5000 pg/ul in water) .
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Set the timer to 1 min.
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Place the chip horizontally in vortex (bioanalyzer room) and votex for 1 min at 2400 rpm.
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Place the chip into the Bioanalyzer and make sure that the run is started within 5 minutes.