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Bionalyzer Agilent HS DNA kit Protocol
Using : Agilent HS DNA kit (Agilent : ref : 5067-4626)
You can download the full protocol here.
Keep all reagents and reagent miwes refrigerated at 4°C when not in use to avoid poor results caused by reagent decomposition. Protect the dye anf dye mixture from light.
Preparing the Gel-Dye Mix
1. Allow the blue-capped High Sensitivity DNA dye concentrate (blue ) and red-capped High Sensitivity DNA gel matrix (red ) to equilibrate to room temperature for 30 minutes.
2. Vortex the blue-capped vial with High Sensitivity DNA dye concentrate (blue ) for 10 seconds and spin down. Make sure the DMSO is completely thawed.
3. Pipette 15 μl of the blue capped dye concentrate (blue ) into a red-capped High Sensitivity DNA gel matrix vial (red ). Store the dye concentrate at 4 °C in the dark again.
4. Cap the tube, vortex for 10 seconds. Visually inspect proper mixing of gel and dye.
5. Transfer the complete gel-dye mix to the top receptacle of a spin filter.
6. Place the spin filter in a microcentrifuge and spin for 10 minutes at room temperature at 2240 g ± 20 % (for Eppendorf microcentrifuge, this corresponds to 6000 rpm).
7. Discard the filter according to good laboratory practices. Label the tube and include the date of preparation.
The prepared gel-dye mix is sufficient for 5 chips. Use the gel-dye within 6 weeks of preparation.
Bioanalyzer preparation
1. Turn on the computer into the bioanalyzer room : agilent session ; password : banalyze.
2. Open the program.
Chip Priming station
1. Take the priming station on the Bioanalyzer room.
2. Remove the plastic cap of the syringe and insert it into 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.
Loading the Gel-Dye mix
1- Allow the gel-dye mix to equilibrate to room temperature for 30 minutes before use. Protect the gel dye mix from light during this time.
2- Take a new High Sensitivity DNA chip out of its sealed bag and place the chip on the chip priming station. 3- Pipette 9.0 μl of the gel-dye mix at the bottom of the well marked G and dispense the gel-dye mix.
4- Set the timer to 60 seconds, make sure that the plunger is positioned at 1 ml and then close the chip priming station. The lock of the latch will click when the Priming Station is closed correctly.
5- Press the plunger of the syringe down until it is held by the clip.
6- Wait for exactly 60 seconds and then release the plunger with the clip release mechanism.
7- Visually inspect that the plunger moves back at least to the 0.3 ml mark.
8- Wait for 5 seconds, then slowly pull back the plunger to the 1 ml position.
9- Open the chip priming station. 10- Pipette 9.0 μl of the gel-dye mix in each of the wells marked G.
Loading the marker
1- Pipette 5 μl of green-capped High Sensitivity DNAmarker (green) into the well marked with the ladder symbol and into each of the 11 sample wells.
Do not leave any wells empty.
Loading the ladder and samples
1- Pipette 1 μl of the yellow-capped High Sensitivity DNA ladder vial (yellow ) in the well marked with the ladder symbol .
2- In each of the 11 sample wells pipette 1 μl of sample (used wells) or 1 μl of marker (unused wells).
3- Set the timer to 60 seconds.
4- Place the chip horizontally in the adapter of the IKA vortex mixer and make sure not to damage the buldge that fixes the chip during vortexing.
5- Vortex for 60 seconds at 2400 rpm.
6- Refer to the next topic on how to insert the chip in the Agilent 2100 Bioanalyzer. Make sure that the run is started within 5 minutes.
Inserting a Chip in the Agilent 2100 Bioanalyzer
1- Open the lid of the Agilent 2100 Bioanalyzer.
2- Check that the electrode cartridge is inserted properly and the chip selector is in position (1).
3- Place the chip carefully into the receptacle. The chip fits only one way.
4- Carefully close the lid. The electrodes in the cartridge fit into the wells of the chip.
5- The 2100 Expert software screen shows that you have inserted a chip and closed the lid by displaying the chip icon at the top left of the Instrument context.
Starting the Chip Run
1- In the Instrument context, select the appropriate assay from the Assay menu.
2- Accept the current File Prefix or modify it.
Data will be saved automatically to a file with a name using the prefix you have just entered. At this time you can also customize the file storage location and the number of samples that will be analyzed.
3- To enter sample information like sample names and comments, complete the sample name table.
4- Click the Start button in the upper right of the window to start the chip run. The incoming raw signals are displayed in the Instrument context.
5- After the chip run is finished, remove the chip from the receptacle of the bioanalyzer and dispose it according to good laboratory practices.
Cleaning Electrodes after a High Sensitivity DNA Chip Run
When the assay is complete, immediately remove the used chip from the Agilent 2100 Bioanalyzer and dispose it according to good laboratory practice. After a chip run, perform the following procedure to ensure that the electrodes are clean (no residues are left over from the previous assay).
1- Slowly fill one of the wells of the electrode cleaner with 350 μl deionized
analysis-grade water.
2- Open the lid and place the electrode cleaner in the Agilent 2100 Bioanalyzer.
3- Close the lid and leave it closed for about 10 seconds.
4- Open the lid and remove the electrode cleaner.
5- Wait another 10 seconds to allow the water on the electrodes to evaporate before closing the lid.