RNA Purification Tutorial - Plasma/Serum RNA

Описание: In this tutorial, we go over the step-by-step workflow for Norgen’s Plasma/Serum RNA Purification Midi Kit (cat. # 56100).

To learn more about this kit, please visit our website https://norgenbiotek.com/product/plas...

To avoid contamination of samples from work surfaces and instruments used, sterilization of the area using 1% bleach followed by wiping down with 70% ethanol is recommended prior to starting.

Preparing a biohazard bag for removal of wastes such as excess plasma/serum and associated tips, tubes and other wastes is recommended in compliance with health and safety regulations.

In this tutorial, a sample input of 250 µL to 1.5 mL will be used.

Step 1. Place 1.5 mL of plasma/serum sample in a 15 mL tube (provided by the user) and add 4.5 mL of Lysis Buffer A. Mix well by vortexing for 10 seconds.

Step 2. Add 3 mL of 100% Isopropanol (provided by the user). Mix well by vortexing for 10 seconds.

Step 3. Transfer 4.5 mL of the mixture from Step 2 into a Midi Spin column assembled with one of the provided collection tubes. Centrifuge for 3 minutes at 1,000 x g (~2,200 RPM). Discard the flowthrough and reassemble the spin column with its collection tube. (Note: Make sure that the lid of the tubes is not tightly closed during centrifugation).

Step 4. Repeat Step 3 one more time until all the mixture from Step 2 has been transferred to the Midi Spin column.

Step 5. Apply 3 mL of Wash Solution A to the column and centrifuge for 3 minutes at 1,000 x g (~2,200 RPM). Discard the flowthrough and reassemble the spin column with its collection tube. (Note: Make sure that lid of the tubes is not tightly closed during centrifugation).

Step 6. Repeat step 5 one more time, for a total of two washes.

Step 7. Spin the column, empty, for 3 minutes at 2,000 x g (~3,000 RPM). Discard the collection tube.

Step 8. Transfer the Midi Spin column to a fresh 15 mL tube (not provided). Apply 400 µL of Elution Buffer F to the column and let stand at room temperature for 2 minutes. Centrifuge for 2 minutes at 500 x g (~1,600 RPM).

Step 9. Reload the eluted RNA from Step 8 back to the Midi Spin column and let stand at room temperature for 2 minutes. Centrifuge for 2 minutes at 500 x g (~1,600 RPM).

Step 10. To the elution from Step 9, add 300 µL of Lysis Buffer A and mix well by vortexing for 10 seconds.

Step 11. Add 400 µL of 96-100% Ethanol (provided by the user). Mix well by vortexing for 10 seconds.

Step 12. Transfer 700 µL of the mixture from Step 11 into a Mini Spin column assembled with one of the provided collection tubes. Centrifuge for 2 minutes at 3,300 x g (~6,000 RPM). Discard the flowthrough and reassemble the spin column with its collection tube.

Step 13. Repeat Step 12 one more time to transfer the remaining mixture into the Mini Spin column.

Step 14. Apply 400µL of Wash Solution A to the column and centrifuge for 1 minute at 3,300 x g (~6,000 RPM). Discard the flowthrough and reassemble the spin column with its collection tube.

Step 15. Repeat Step 14 two more times, for a total of three washes.

Step 16. Spin the column, empty, for 2 minutes at 13,000 x g (~14,000 RPM). Discard the collection tube.

Step 17. Transfer the spin column to a fresh 1.7 mL Elution tube. Apply 50 µL of Elution Solution A to the column and let stand at room temperature for 2 minutes. Centrifuge for 1 minute at 400 x g (~2,000 RPM), followed by 2 minutes at 5,800 x g (~8,000 RPM).

Step 18. For maximum recovery, transfer the eluted buffer back to the column and let stand at room temperature for 2 minutes. Centrifuge for 1 minute at 400 x g (~2,000 RPM), followed by 2 minutes at 5,800 x g (~8,000 RPM).

Plasma/Serum RNA is ready for the downstream application of your choice. For an explanation of expected yields and recommendations for quantification of the RNA, please read the protocol on our website https://norgenbiotek.com/product/plas...

If you have questions or would like to place an order, give us a call at 1-866-NORGENB (Toll-free) or email our product specialists at [email protected]

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