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ARK Genomics
Standard Operating Procedure
S.O.P#
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: | IGF005.00 | |
Title
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: | Use of the Agilent 2100 Bioanalyser for RNA quality control | |
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| 1.0 | BACKGROUND
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| 2.0 | PURPOSE
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| 3.0 | MATERIALS | ||
RNA 6000 Nano assay kit |
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Vortex mixer
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| 4.0 | DECONTAMINATION OF ELECTRODES | ||
The RNAse decontamination procedure must be carried out on a daily basis when the Bioanalyser is in use. |
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| 4.1 | Slowly fill the bottom right well of an electrode cleaner with 350ml RNAseZAP and label. | ||
| 4.2 | Open the lid and place the electrode cleaner in the Agilent 2100 Bioanalyser. Close the lid and leave for one minute. | ||
| 4.3 | Open the lid and remove the electrode cleaner. Rinse out the RNAseZAP chip at the end of every day. | 4.4 | Slowly fill the bottom right well of the other electrode cleaner chip with 350ml RNAse-free water, label and place in the Agilent 2100 Bioanalyser. |
| 4.5 | Close the lid then, after ten seconds remove the electrode cleaner chip. Wait another ten seconds for the water to evaporate.
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| 5.0 | CHIP PREPARATION - Edition April 2003 |
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Always keep exposure to light a minimum and
allow to equilibrate to room temperature for at least 30 minutes prior
to use. |
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| 5.1 | Prepare the gel matrix by placing 550ml of RNA 6000 Nano gel matrix (red cap) into a spin filter. Centrifuge at 1500 x g for 10 minutes. | ||
| 5.2 | Aliquot 65ml of the filtered gel into 0.5ml RNase free microfuge tubes provided with the kit. The filtered gel matrix must be used within 4 weeks, label the microfuge tubes with the date of preparation. | ||
| 5.3 | To prepare the gel-dye mix vortex the RNA 600 Nano dye concentrate (blue cap) for 10 seconds, spin down and add 1ml of the dye into a 65ml aliquot of the filtered gel matrix. | 5.4 | Vortex the gel-dye mix well and then centrifuge the tube at 13000 x g for 10 minutes. |
| 5.5 | Take a new RNA chip out of its sealed bag and place on the chip priming station. Make sure the clip of the chip priming station is set to its upper position (RNA) | ||
| 5.6 | Using a P20, aspirate 9ml of the gel-dye mix and pipette into well G (Black) taking care to dispense into the bottom of the well holding the pipette vertically. | ||
| 5.7 | Make sure the syringe plunger of the chip priming station is set to 1ml before closing the lid with a click. | ||
| 5.8 | Push the plunger down so that it rests underneath the clip and leave for 30 seconds before releasing the clip. The plunger will travel back up the barrel, however pull it back to 1ml if it does not reach it by itself. | ||
| 5.9 | Open the chip priming station, remove the chip, and check for any air bubbles. If there are any bubbles repeat step 5.6 – 5.7.
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| 6.0 | LOADING THE CHIP |
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| 6.1 | Log onto the Agilent PC using the username Administrator and password 3000hanover and double click on the Agilent 2100 biosizing icon to open the software. | ||
| 6.2 | Select assay type by choosing assay on the menu and selecting RNA. This will give a choice of assay types. Choose either total RNA of mRNA assay. | ||
| 6.3 | Pipette 9ml of the gel-dye mix into the other two wells marked G, and 5ml of the RNA 6000 Nano marker (green cap) into the well marked with a ladder and into each of the twelve sample wells. | 6.4 | Add 1ml of RNA 6000 ladder into the well marked with a ladder. |
| 6.5 | Pipette 1ml of each sample into each of the sample wells. If there are any unused sample wells add another 1ml of RNA 6000 Nano marker. Place the chip on the IKA vortexer and vortex for one minute. | ||
| 6.6 | Place the loaded chip into the Bioanalyser. The display will show an RNA chip. | ||
| 6.7 | Press on the word Start above the picture of the Bioanalyser. Enter the name of the assay in the window which appears. | ||
| 6.8 | After the chip has run remove the chip and clean the electrodes with the RNAse free water cleaning chip for ten seconds. | ||