Total RNA Purification Micro Kit Tutorial (Cat. 35300)

In this tutorial, you will learn how to use the Total RNA Purification Micro Kit (Cat. 35300) to rapidly purify total RNA, including microRNA, from small input amounts.

To learn more about this kit and view the complete protocol, please visit our website https://norgenbiotek.com/product/tota...

Upon opening this kit you will find:
A detailed product insert, Micro spin columns, Collection tubes, Elution tubes, Wash solution A, Buffer RL, Elution Solution A.

1:02 To lyse your sample:
1. Transfer cell suspension to an RNase-free tube and centrifuge at no more than 425 x g (~2,000 RPM) for 10 minutes to pellet cells.
2. Carefully decant the supernatant.
3. 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.
4. Add 200 µL of 96 - 100% Ethanol to the lysate. Mix by vortexing for 10 seconds.

1:40 Next, bind the RNA to the column:
1. Assemble a micro spin-column with one of the provided collection tubes.
2. Apply up to 600 µL of the lysate with the ethanol from the previous step onto the column and centrifuge for 1 minute at 3,500 x g (~6,000 RPM).
3. Discard the flowthrough. Reassemble the spin column with its collection tube.
4. Depending on your lysate volume, repeat the previous two steps as necessary.

2:13 Now, wash the column of all unwanted nucleic acids:
Before you begin the wash steps, add 90 mL of 100% ethanol to the supplied bottle containing the Wash Solution A. This will give a final volume of 128 mL. The label on the bottle has a box that may be checked to indicate that the ethanol has been added

1. Apply 400 µL of Wash Solution A to the column and centrifuge for 1 minute.
2. Discard the flowthrough and reassemble the spin column with its collection tube.
3. Repeat the wash steps two more times, for a total of three washes.
4. Spin the column for 2 minutes in order to thoroughly dry the resin. Discard the collection tube.

2:59 Finally, we will elute the RNA:
1. Place the column into a fresh 1.7 mL Elution tube provided with the kit.
2. Add 40 µL of Elution Solution A to the column.
3. Centrifuge for 2 minutes at 425 x g (~2,000 RPM), followed by 1 minute at 20,800 x g (~14,000 RPM) Note the volume eluted from the column.

The purified RNA sample may be stored at –20°C for a few days. It is recommended that samples be placed at –70°C for long term storage.

Downstream Applications
RNA Purified using this kit can be used in the following downstream applications:
Next Generation Sequencing, RT-qPCR, Viral Detection, Microarrays, Northern Blotting and More!

Or, give us a call at 1-866-NORGENB (Toll-free) or email our product specialists at [email protected].

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