A method to prepare Sera-Mag SpeedBeads for purification and size selection of nucleic acids

Place a 50 ml conical tube on a balance and tare the balance.

Slowly add 5.475 g of Tween 20 to the conical tube using a serological pipette.

Remove the conical tube from the balance and add 45 ml of MilliQ water.

Thoroughly mix the solution with shaking until homogenous (approximately 20 min). Store 10% (v/v) Tween 20 at room temperature.

Prepare the following buffer in a 50 ml conical tube:

Speedbead DNA buffer

A	B	C
Reagent	Volume	Final conc.
1 M Tris-HCl (pH 8.0)	500 μl	10 mM
0.5 M EDTA (pH 8.0)	100 μl	1 mM
10% (v/v) Tween 20	250 μl	0.05%
Nuclease-free water	49.15 ml	–
Total	50 ml	–

Equilibrate Sera-Mag Speedbeads to room temperature and mix thoroughly by vortexing.

Immediately after mixing, transfer 1 ml of bead slurry to a 1.5 ml microcentrifuge tube. The speedbeads will settle quickly. The bead slurry must be thoroughly mixed immediately before transferring the 1 ml to a 1.5 ml microcentrifuge tube.

Place samples on a magnetic stand until solution is clear.

Carefully remove and discard cleared supernatant without disturbing the bead pellet.

Remove beads from magnetic stand. Add 1 ml of speedbead DNA buffer and vortex beads on setting 4 for 15 seconds. Very briefly centrifuge samples after mixing. Stop the centrifuge before beads settle.

Repeat Steps 8-10 twice. Keep bead slurry at room temperature.

Prepare the following mixture in a 50 ml conical tube as described in Steps 13-21:

Speedbead DNA binding mix

A	B	C
Reagent	Volume	Final conc.
PEG-8000	9 g	18% (w/v)
5 M NaCl	100 μl	2 M
1 M Tris-HCl (pH 8.0)	500 μl	10 mM
0.5 M EDTA (pH 8.0)	100 μl	1 mM
10% (v/v) Tween 20	250 μl	0.05%
Prepared speedbeads	1 ml	–
Nuclease-free water	up to total	–
Total	50 ml	–

Add 9 g of PEG-8000 to a 50 ml conical tube.

Add 20 ml of 5 M NaCl to the conical tube.

Add 500 μl of 1.0 M Tris-HCl (pH 8.0) to the conical tube.

Add 100 μl 0.5 M EDTA (pH 8.0) to the conical tube.

Add nuclease-free water (up to 45 ml) to the 50 ml conical tube.

Mix the solution with shaking until PEG-8000 goes into solution (approximately 5-10 min).

Add 250 μl of 10% (v/v) Tween 20 to the conical tube and mix with gentle shaking until Tween 20 goes into solution (approximately 5 min).

Ensure beads (prepared in Steps 6-11) are fully resuspended in speedbead DNA buffer and add the bead suspension to the conical tube.

Add nuclease-free water up to 50 ml to the conical tube and mix the speedbead DNA binding mix thoroughly by vortexing. Nuclease-free water can be added up to 50 ml by eye using a 1000 μl pipet.

Wrap the speedbead DNA binding mix in foil and store at 4°C. Speedbead DNA binding mix can be stored at 4°C for several months.

Validate speedbeads after preparation to ensure they are working as expected (see Step 25).

Prepare the following buffer in a 50 ml conical tube:

Speedbead DNA binding buffer

A	B	C
Reagent	Volume	Final conc.
PEG-8000	9 g	18% (w/v)
5 M NaCl	100 μl	2 M
1 M Tris-HCl (pH 8.0)	500 μl	10 mM
0.5 M EDTA (pH 8.0)	100 μl	1 mM
10% (v/v) Tween 20	250 μl	0.05%
Nuclease-free water	up to total	–
Total	50 ml	–

Prepare 10 aliquots of DNA ladder in 0.2 ml PCR tubes. Combine 3 μl of a 1:10 dilution of DNA ladder and 22 μl of 10 mM Tris-HCl (pH 8.0) for each aliquot if using Quick-load purple 1 kb plus DNA ladder.

Add 0.4X, 0.5X, 0.6X, 0.8X, 1.0X, 1.2X, 1.4X, 1.6X, 1.8X, and 2.0X volumes of speedbeads to the aliquots of DNA ladder. Careful and consistent pipetting of speedbeads is essential to ensure accurate size selection. Additional ratios of beads to sample can be tested as needed.

Purify the aliquots of DNA ladder as described in the general method for speedbead purification .

Elute DNA in 21 μl of 10 mM Tris-HCl (pH 8.0) prewarmed to 50°C.

Combine 4 μl of 6X DNA loading dye with each purified sample (20 μl). Run 8 μl of the mixture on a 1.0% agarose gel at 150 V for 60 min. As a control, run 8 μl of a mixture of DNA ladder (3 μl of a 1:10 dilution), 10 mM Tris-HCl (pH 8.0) (17 μl), and 6X DNA loading dye (4 μl). Conditions for gel electrophoresis (e.g., percent agarose, voltage, time) can vary depending setup.

Equilibrate speedbeads to room temperature and mix thoroughly.

Carefully add indicated amount of speedbeads to the sample.

Mix well by pipetting up and down a minimum of 10 times. Alternatively, vortex samples on setting 4 for 3-5 seconds. If samples require centrifugation after mixing, stop the centrifuge before beads settle.

Incubate samples at room temperature for 5 min. Prolonged incubation or incubation at low temperature will increase binding of small DNA fragments (e.g., adapter dimers).

Place samples on a magnetic stand for 5 min (or until solution is clear) to collect beads. Keep samples on the magnetic stand for Steps 34-38.

Carefully remove and discard cleared supernatant without disturbing bead pellet.

Add 200 μl of 80% ethanol (freshly prepared with MilliQ water) and incubate for 30 s. Carefully remove ethanol and discard without disturbing bead pellet.

Repeat Step 36 once. Carefully inspect samples and remove remaining ethanol using a 10 μl pipet.

Air dry samples for 2 min with lid open. Do not over-dry samples. Speedbeads (and other SPRI products) will clump and can not be fully resuspended during elution if samples are over-dried.

Remove samples from the magnetic stand. Add the indicated volume of appropriate solution for elution and mix well by pipetting or vortexing (see Step 32).

Incubate mixture at room temperature for 2-5 min.

Place samples on a magnetic stand for 2 min (or until solution is clear) to collect beads.

Transfer cleared supernatant (volume of solution added for elution less 1 μl) to a fresh PCR tube.