ChIP-chip
Details

Bionalyzer pico kit

Material :

  • Rnase-free water

  • Pipettes

  • 0,5 ml tubes Rnase free

  • Heating block

  • Pico kit and reagents

 

Primer preparation (Only before the first run)

  1. Spin ladder down.

  2. Heat denature the ladder for 2 min at 70 °C

  3. Immediately cool down the vial on ice

  4. Add 90 uL of Rnase-free water and mix thoroughly

  5. Prepare aliquots of ladder

  6. Store aliquots at -70°C (see « Pico ladder » tubes at -70°C)

  7. Before use, thaw ladder aliquots and keep them on ice.

 

Chip Priming station

  1. Take the priming station on the Bioanalyzer room

  2. Inser your syringe from the clip.

  3. Slide it into the hole of the luer lock adapter and screw it tightly to the chip priming station.

  4. Open the chip priming station by pulling the latch

  5. Using a screwdriver, open the screw at the underside of the base plate.

  6. Lift the base plate and insert it again in position C. Retighten the screw.

  7. 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)

  1. Fill one of the wells of the chip cleaner with 350 uL of fresh Rnase-free water

  2. Turn on the computer into the bioanalyzer room : agilent session ; password : banalyze.

  3. Open the program and open the lid of the analyzer.place the electrode cleaner in the Agilent 2100 Bioanalyzer

  4. 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).

  5. Open the lid and remove the electrode cleaner ( ! there is only one cleaner chip so keep it for future use).

  6. 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 )

  1. Allow all reagents to equilibrate to room temperature for 30 minutes before use.

  2. Place 550 ul of RNA 6000 pico gel matrix (red cap) into the top receptacle of a spin filter.

  3. Spin the filter tube for 10 min at 4000 rpm

  4. 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

  1. Allow all reagents to equilibrate to room temperature for 30 minutes befor use. Protect the dye concentrate from light.

  2. Vortex RNA 6000 Pico dye concentrate (blue cap) for 10 sec and spin down.

  3. Add 1 uL of RNA 6000 Pico dye concentrate (blue cap) to 65 uL aliquot of filtered gel.

  4. Vortex thoroughly and store the dye concentrate at 4 °C in the dark again.

  5. 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

  1. Take a new RNA pico chip out of its sealed bag.

  2. Place the chip on the chip priming station.

  3. 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.

  4. Set the timer to 30 seconds, make sure that the plunger is positioned at 1 ml and then close the chip

  5. priming station. The lock of the latch will click when the chip priming station is closed correctly.

  6. Press the plunger of te syrunge down until it is held by the clip.

  7. Wait for exactly 30 sec and release the plunger moves back at least to the 0,3 mL mark.

  8. Wait for 5 sec then slowly pull back the plunger to the 1 ml position.

  9. Open the chip priming station.

  10. Pipette 9 uL of the gel dye mix in each of the wells marked G

 

Loading solution and marker

  1. Pipette 9 uL of the RNA 6000 pico conditioning solution (white cap) into the well marked CS

  1. 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.

  1. To minimize secondary structure, you may heat denature 70°C for 2 min the samples before loading them on the chip.

  2. Pipette 1 ul of the diluted RNA 6000 pico Ladder into the well marked with the ladder sybol (DNA molecule symbol) .

  3. Pipette 1 uL of each sample into each of the 11 sample wells (RNA concentration accepted: 50-5000 pg/ul in water) .

  4. Set the timer to 1 min.

  5. Place the chip horizontally in vortex (bioanalyzer room) and votex for 1 min at 2400 rpm.

  6. Place the chip into the Bioanalyzer and make sure that the run is started within 5 minutes.