Total RNA Purification Tutorial (Cat. 17200)

In this tutorial, you will learn how to use one of Norgen Biotek’s Best-Selling-Kits, the Total RNA Purification Kit (Cat. 17200), for the rapid purification of total RNA.
norgenbiotek.com/product/tota...
Cell Lysate from Different Sample Types
00:01:37 #1 - Cell Lysate Preparation from Cells Growing in Suspension and Lifted Cell
- Transfer cell suspension to an RNase-free tube (not provided) and centrifuge at no more than 425 x g (~2,000 RPM) for 10 minutes to pellet cells.
- Carefully decant the supernatant. A few µL of media may be left behind with the pellet in order to ensure that the pellet is not dislodged.
- Add 350 µL of Buffer RL to the pellet. Lyse cells by vortexing for 15 seconds. Ensure that the entire pellet is completely dissolved before proceeding to the next step.
- Add 200 µL of 96 - 100% ethanol (provided by the user) to the lysate. Mix by vortexing for 10 seconds.
00:02:30 #2 - Cell Lysate Preparation from Animal Tissues
- Excise the tissue sample from the animal.
- Determine the amount of tissue by weighing. We recommend starting with an input of no more than 10 mg.
- Transfer the tissue into a mortar that contains an appropriate amount of liquid nitrogen to cover the sample. Grind the tissue thoroughly using a pestle.
- Allow the liquid nitrogen to evaporate, without allowing the tissue to thaw.
- Add 600 µL of Buffer RL to the tissue sample and continue to grind until the sample has been homogenized. Homogenize by passing the lysate 5-10 times through a 25-gauge needle attached to a syringe.
- Using a pipette, transfer the lysate into an RNase-free microcentrifuge tube (not provided).
- Spin the lysate for 2 minutes to pellet any cell debris. Transfer the supernatant to another RNase-free microcentrifuge tube. Note the volume of the supernatant/lysate.
- Add an equal volume of 70% ethanol to the lysate volume collected (100 µL of ethanol is added to every 100 µL of lysate). Vortex to mix.
00:03:52 #3 - Cell Lysate Preparation from Blood
- Transfer up to 100 µL of non-coagulating blood to an RNase-free microcentrifuge tube (not provided).
- Add 350 µL of Buffer RL to the blood. Lyse cells by vortexing for 30 seconds. Ensure that the mixture becomes a light red colour before proceeding to the next step.
- Add 200 µL of 96 - 100% ethanol (provided by the user) to the lysate. Mix by vortexing for 10 seconds.
00:04:31 #4 - Cell Lysate Preparation from Viral Suspension
- Transfer up to 100 µL of viral suspension to an RNase-free microcentrifuge tube (not provided).
- Add 350 µL of Buffer RL. Lyse viral cells by vortexing for 15 seconds. - Ensure that the mixture becomes transparent before proceeding to the next step.
- Add 200 µL of 96 - 100% ethanol (provided by the user) to the lysate. Mix by vortexing for 10 seconds.
Now that the samples have been lysed, we will complete the purification of RNA from our sample types.
Total RNA Purification from All Types of Lysate
00:05:16 Binding RNA to Column
- Assemble a column with one of the provided collection tubes.
Apply up to 600 µL of the lysate with the ethanol from the previous step onto the column and centrifuge for 1 minute at 20,800 x g (~14,000 RPM).
Discard the flowthrough and reassemble the spin column with its collection tube.
Depending on your lysate volume, repeat until the desired volume is reached.
00:05:45 Column Wash
- Apply 400 µL of Wash Solution A to the column and centrifuge for 1 minute.
- Discard the flowthrough and reassemble the spin column with its collection tube.
- Wash the column two more times with 400 µL of Wash Solution A and centrifuging for one minute, for a total of three washes.
- Spin the column empty for 2 minutes in order to thoroughly dry the resin. Discard the collection tube.
00:06:22 RNA Elution
- Place the column, after being dried, into a fresh 1.7 mL Elution tube provided with the kit.
- Add 50 µL of Elution Solution A to the column. We do not recommend eluting the RNA in a lower elution volume.
- Centrifuge for 2 minutes at 425 x g (~2,000 RPM), followed by 1 minute at 20,800 x g (~14,000 RPM).
The purified RNA is now ready to be used or stored at -70* C for long-term storage.
Learn more about this kit and view the complete protocol on our website norgenbiotek.com/product/tota...
Or, give us a call at 1-866-NORGENB (Toll-free) or email our product specialists at [e-mail].
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[@MICROVISIONDETECTIONS]

Excellent ! microMan

[@manubp00]

Hello! I the first step, can I leave my sample at the RL buffer for how long? or is´t not recommended?