Plasma/Serum Cell-Free Circulating DNA Purification Tutorial (Cat. 55100)

In this tutorial, we go over the step-by-step workflow for Norgen’s Plasma/Serum Cell-Free Circulating DNA Purification Mini Kit (cat. # 55100).
To learn more about this kit and view the most up to date protocol, please visit our website norgenbiotek.c....
(Please note there is a slight discrepancy between this tutorial and the most updated protocol available on the website.)
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.
Step 1: Preheat a water bath or heat block to 60 degrees Celsius and vortex the proteinase K before use.
Step 2: Place 500 µL of plasma/serum sample into a 2 ml tube provided by the user. Add 30 microliters of Proteinase K and mix well by vortexing for 10 seconds. Then, incubate at room temperature for 10 minutes.
Step 3: After incubation, add 1 mL of Binding Buffer B and mix well by vortexing for 10 seconds.
Step 4: Transfer 750 µL of the mixture from the previous step into a mini spin column assembled with one of the provided collection tubes. Centrifuge for 2 minutes at 3,800 g (~6,000 RPM). Discard the flowthrough and reassemble the spin column with its collection tube.
Step 5: Repeat this step to transfer the remaining mixture into the mini spin column.
Step 6: Apply 500 µL of solution WN to the column and centrifuge for 1 minute at 3,800 g (~6,000 RPM). Discard the flowthrough and reassemble the spin column with its collection tube.
Step 7: Apply 500 µL of Wash Solution A to the column and centrifuge for 1 minute at 3,800 g (~6,000 RPM). Discard the flowthrough and reassemble the spin column with its collection tube. Repeat this step for a total of 2 washes.
Step 8: Spin the column, empty, for 3 minutes at 20,800 g (~14,000 RPM). Discard the collection tube.
Step 9: Transfer the spin column to a fresh 1.7 mL Elution tube. Apply 100 µL of Elution Buffer B to the column and let stand at room temperature for 2 minutes. Centrifuge for 2 minutes at 420 g (~2,000 RPM), followed by 2 minutes at 6,800 g (~8,000 RPM).
Step 10: Add 300 µL of Binding Buffer B to the 100 µL of eluted DNA from the previous step, and mix well by vortexing for 10 seconds.
Step 11: Transfer the entire mixture from the previous step to a Micro Spin column assembled with one of the provided collection tubes. Centrifuge for 1 minute at 3,800 g (~6,000 RPM). Discard the flowthrough and reassemble the spin column with its collection tube.
Step 12: Apply 500 µL of Wash Solution A to the column and centrifuge for 1 minute at 3,800 x g (~6,000 RPM). Discard the flowthrough and reassemble the spin column with its collection tube.
Step 13. Repeat this step one more time, for a total of two washes.
Step 14. Spin the column, empty, for 3 minutes at 20,800 g (~14,000 RPM). Discard the collection tube.
Step 15. Transfer the spin column to a fresh 1.7 mL Elution tube. Apply 25-50 µL of Elution Buffer B to the column and let stand at room temperature for 2 minutes. Centrifuge for 1 minute at 420 g (~2,000 RPM), followed by 2 minutes at 6,800 x g (~8,000 RPM).
The plasma/serum DNA is now ready for the downstream application of your choice! For an explanation of the expected yields and recommendations for quantification of the DNA, please read the protocol on our website - norgenbiotek.c....
To learn more about this kit or our other cell-free DNA Purification Kits, check out our website norgenbiotek.c....
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 [e-mail]
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