Quick Protocol
PAGE 1
•
PART# FB238
Additional protocol information in Technical Manual #TM673, available online at:
www.promega.com
Instructions for Use of Product AS1840.
Maxwell
®
RSC ccfDNA LV Plasma Kit
This Quick Protocol provides instructions for use of the Maxwell
®
RSC ccfDNA LV Plasma Kit (Cat.# AS1840) with Maxwell
®
Instruments to purify circulating cell-free DNA (ccfDNA) from plasma samples. For detailed instructions, including information on
instrument setup and troubleshooting, please refer to the
Maxwell
®
RSC ccfDNA LV Plasma Kit Technical Manual
#TM673, available at:
www.promega.com/protocols/
Preparing Plasma Samples
Materials to Be Supplied by the User
• whole blood or plasma
• benchtop centrifuge
Whole blood should be processed immediately after collection or stored at +2°C to +10°C until plasma preparation. Centrifuge whole
blood from EDTA tubes for 10 minutes at 2,000 ×
g
to pellet the red and white blood cells. For stabilized blood collection tubes, follow
the manufacturer’s instructions.
After either EDTA or stabilized blood collection tubes are first centrifuged, use a pipette to carefully remove as much plasma as
possible without disturbing the buffy coat. To ensure that no white blood cells are transferred, centrifuge the plasma a second time for
10 minutes at 2,000 ×
g
, and transfer the supernatant to a clean tube.
Store plasma at +2°C to +10°C for up to 1 week. For longer storage times, store plasma at –10˚C to –30˚C (or below –65˚C). Avoid
exposing plasma to freeze-thaw cycles.
Manual Sample Preprocessing Using a Rotisserie Shaker
1. Add 2–8ml of plasma to a 15ml or 50ml tube. Add an equal volume of Binding Buffer.
2. Shake the bottle containing the Maxwell
®
Resin E until it is
completely
resuspended.
3. Add 100µl of magnetic resin.
4. Incubate for 45 minutes while shaking. We recommend a rotisserie shaker; the resin must be kept in suspension for the entire incubation.
5. Centrifuge the tubes at 1,000 ×
g
for 2 minutes to pellet the resin. Alternatively, a magnetic stand can be used to immobilize the resin.
6. Carefully decant the supernatant. While decanting, we recommend placing a magnet alongside the resin pellet in the tube to fix it in
place. Proceed to the section Automated ccfDNA Purification.
Preprocessing Samples with Heater Shaker Magnet Instrument (HSM 2.0)*
1. Add 2–8ml of plasma to a 50ml tube. Add an equal volume of Binding Buffer.
2. Shake the bottle containing the Maxwell
®
Resin E until it is completely resuspended.
3. Add 100µl of magnetic resin to each tube.
4. Place the tube(s) in the HSM.
5. Open the Promega HSM 2.0 Application Software and select
Start Protocol
.
6. A window will open to select the method. Double-click the “HSM 2.0 RSC ccfDNA LV Plasma v1.0.0.nsp” tile or select the file and
choose
Open
to launch the method.
7. The ‘Select Available HSM 2.0 Instrument’ window will open. Select the HSM 2.0 unit that will run the method and choose
OK
.
8. The protocol window will launch. Press
Start
and follow the instructions in the software.
9. The HSM will shake for 45 minutes and then stop. The magnets will engage, drawing the resin to the side of the tubes.
10. When the resin is completely magnetized, use a pipette to remove the supernatant. Remove the tubes from the HSM. Proceed to
the section Automated ccfDNA Purification.
*HSM 2.0 Instrument (Cat.# A2715) must be purchased separately.
