RNA Extraction using automated Promega Maxwell RSC Instrument
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- Опубликовано: 23 авг 2024
- Automated RNA Extraction using Maxwell RSC instrument offers easy and convenient way of RNA extraction. All you have to do is carry out sample preparation and load the samples in the cartridges and start the extraction process. It takes about 52 minutes from this point to complete the extraction process.
I will upload a separate video to discuss how good is the RNA quality.
Kit Content:
1-Thioglycerol
Maxwell® RSC Cartridge (RSCD)
Elution Tubes (0.5ml)
Blue Dye
Lysis Buffer
Nuclease-Free Water
Homogenization Solution
DNase I (lyophilized)
RSC Plungers
Preparation of solutions and cartridge:
1-Thioglycerol/Homogenization Solution:
A volume of 200 µl of 1-Thioglycerol/Homogenization Solution is needed for each sample.
To prepare a working solution:
1-Thioglycerol ……………………….... 20 µl
Homogenization Solution ………… to 1 ml
1-Thioglycerol is viscous, so careful pipetting is required for accurate measurement.
Alternatively, add 600 µl of 1-Thioglycerol to the 30 ml bottle of Homogenization Solution.
Before use, chill the 1-Thioglycerol/Homogenization Solution on ice or at 2-10°C.
Note: Store the 1-Thioglycerol/Homogenization Solution at 2-10°C, where it is stable for up to 30 days.
DNase I Solution:
• Add 275 µl of Nuclease-Free Water to the vial of lyophilized DNase I.
• Invert to rinse DNase off the underside of the cap and swirl gently to mix; do not vortex.
• Add 5 µl of Blue Dye to the reconstituted DNase I as a visual aid for pipetting.
• Dispense the DNase I Solution into single-use aliquots in nuclease-free tubes.
• Store reconstituted DNase I at -30°C to -10°C.
[DNase I solution maintains activity for up to 10 freeze-thaw cycles.]
Maxwell® RSC simplyRNA Cartridge Preparation:
To maintain an RNase-free environment during processing, change gloves before handling cartridges, MaxwellR CSC/RSC Plungers and Elution Tubes.
• Place the cartridges to be used in the deck tray with the printed side facing away from the Elution Tubes.
• Press down on the cartridge to snap it into position.
• Carefully peel back the seal so that all plastic comes off the top of the cartridge.
• Ensure that all sealing tape and any residual adhesive are removed before placing cartridges into the instrument.
Note: If you are processing fewer than 16 samples, centre the cartridges on the deck tray.
• Place a MaxwellR CSC/RSC Plunger in well #8 of each cartridge.
[Well #8 is the well closest to the Elution Tube]
• Place 0.5 ml Elution Tubes in the front of the deck tray.
• Add 50 µl of Nuclease-Free Water to the bottom of each Elution Tube.
Notes:
If Nuclease-Free Water is on the side of the tube, the elution may be suboptimal.
Use only the 0.5 ml Elution Tubes provided in the kit.
Purification of Total RNA from Cell Culture
[Trypsinize adherent cells following normal protocols. Pellet cells at low speed (e.g., 300 × g for 3 minutes)]
1. If the cells are frozen, thaw on ice.
2. Remove medium.
3. Add 200 µl of chilled 1-Thioglycerol/Homogenization Solution to the cell pellet and vortex until pellet is dispersed and cells appear lysed.
[A pipette may be used to disperse pellets before vortexing. Alternatively, cells can be homogenized. Store lysed cells on ice if there is a delay before processing.]
4. Add 200 µl of Lysis Buffer (Part# MC501C) to 200 µl of lysed cells.
5. Vortex vigorously for 15 seconds to mix.
6. Transfer all 400 µl of lysate to well #1 of the MaxwellR RSC Cartridge.
[Well #1 is the closest to the printed side and farthest from the elution tube.]
7. Add 5 µl of blue DNase I Solution to well #4 (yellow reagent).
[After adding the blue DNase I Solution, the solution in well #4 will be green.]
Proceed to Section for instructions on loading samples on the instrument and beginning the automated purification.
Maxwell® RSC Instrument Setup and Run
1. Turn on the MaxwellR RSC Instrument and Tablet PC.
2. Log on to the Tablet PC, and start the RSC software.
The instrument will power up, proceed through a self-check and home all moving parts.
3. Press Start to access the extraction method selection screen.
4. On the extraction method selection screen, select a method using one of the two options below:
o a. Manually touch the RSC simplyRNA Cell or RSC simplyRNA Tissue method.
o b. Use a bar code reader to scan the 2D bar code on the kit box to automatically select the appropriate method.
5. Verify that the appropriate RSC simplyRNA method has been selected, and press the Proceed button.
6. On the cartridge selection screen, touch the cartridge positions to deselect any positions that will not be used for this extraction run.
7. Transfer the deck tray containing prepared cartridges onto the MaxwellR RSC Instrument platform.
Follow on-screen instructions.
Thank you ! Now I know
Do you know what components have each tube of the cartridge?? And which is the function of the Plunger?
The kits cartridges help purify samples using paramagnetic particles, which provide a mobile solid phase to optimize sample capture, washing and purification of nucleic acid. Maxwell Instruments are magnetic particle-handling instruments that efficiently bind nucleic acids to the paramagnetic particle in the first well of a prefilled cartridge. The samples are processed through a series of washes
before nucleic acids are eluted.
@@innovative_learner thanks!!
Thanks for your video. Do you nanodrop these eluates? Have you noted any reagent carry over?
Yes, the RNA following elution goes through quality testing including Nanodrop and Bioanalyzer and I only use RNA if it satisfies all these. Hope that hlps. Thanks.
@@innovative_learner Thank you for your answer. Do you blank the nanodrop with the nuclease free water from the kit? If so what is the 260/230 ration like? There must be a certain amount of carry over from the last well the plunger is in, into the elution tube with H20 in it. I wondered if this might be deliberate to create a buffered solution in the elution tube.
Is there a special reason for mounting cartridges below 16 in the center?
No, there is no specific reason for this. You can insert cartridge anywhere you like. Hope that clarifies. Thank you.
❣️ Promo*SM