TITLE: Rapid, effective and low-cost purification of dideoxy-sequencing reactions by home-made magnetic beads suspension and magnetic separator
AUTHORS: Hidenori Sassa

[DESCRIPTION]
Removal of excess dideoxy terminators from the sequencing mix after the enzymatic reaction is a key process affecting the dideoxy/Sanger sequencing quality. Ethanol precipitation may be the most popular clean-up method because of its low costs; however, it takes a long centrifugation time and frequently results in low quality sequence data. Commercially available clean-up kits provide high quality sequence data, while they generally have high cost. Here, we describe rapid, effective and low-cost dideoxy terminator clean-up method using a home-made magnetic beads suspension, MagNA, and magnetic separator. We found that MagNA enables rapid and efficient clean-up at ~1/100 of the cost of commercially available kits. The magnetic separator made using low-cost neodymium magnets worked well for the MagNA separation, representing a the rapid, efficient and cost-effective dideoxy terminator clean-up system.

[STEPS]
SECTION: MagNA suspension
1. 
SECTION: MagNA suspension
1.1. Prepare 50 ml buffer without magnet beads as follow. Final concentration of each chemical is indicated in parenthesis. 
Dissolve 9 g PEG 8,000 (18 %), 2.92 g NaCl (1 M), 5 ml 10x TE (1x), 25 µl Tween-20 (0.05 %) and 50 µl ProClin 300 (0.1 %) in DW to prepare 50 ml PEG-NaCl buffer.
SECTION: MagNA suspension
1.2. Mix thoroughly Carboxyl-modified Sera-Mag Magnetic Speed-beads (Hydrophobic) (Cytiva, cat. #65152105050250), take 1 ml Sera-Mag suspension and separate the beads by using magnetic separator. Add 1 ml 1x TE, mix, stand on magnetic separator, and remove the supernatant. Repeat this wash once.
SECTION: MagNA suspension
1.3. Add 1 ml PEG-NaCl buffer, mix and transfer the suspension to the PEG-NaCl buffer tube to prepare MagNA suspension. Store at 4 °C at least for one year.
SECTION: Home-made magnetic separator
2. Set pairs of magnets at the edge of the insert by their magnetic force as they line to tip halls (Fig. 1b and c).
SECTION: Clean-up of dideoxy sequencing reaction by MagNA and home-made magnetic separator
3. The procedure is based on the protocol of CleanSeq (Beckman Coulter) as below.
SECTION: Clean-up of dideoxy sequencing reaction by MagNA and home-made magnetic separator
3.1. Shake MagNA to fully resuspend the magnetic beads.
SECTION: Clean-up of dideoxy sequencing reaction by MagNA and home-made magnetic separator
3.2. To the 10 µl sequencing reaction, add 10 µl MagNA suspension.
SECTION: Clean-up of dideoxy sequencing reaction by MagNA and home-made magnetic separator
3.3. Add 42 µl 85 % EtOH (Volume of 85% Ethanol = 2.077  (10 L + Sequencing Sample Volume) and mix thoroughly.
SECTION: Clean-up of dideoxy sequencing reaction by MagNA and home-made magnetic separator
3.4. Place the tube in the home-made magnetic separator for 2~3 min (Fig. 1d).
SECTION: Clean-up of dideoxy sequencing reaction by MagNA and home-made magnetic separator
3.5. Remove the supernatant, add 100 µl of 85 % ethanol, and remove the supernatant after >30 sec. Repeat the step once.
SECTION: Clean-up of dideoxy sequencing reaction by MagNA and home-made magnetic separator
3.6. Remove the tube from the separator, and add 40 µl DW.
SECTION: Clean-up of dideoxy sequencing reaction by MagNA and home-made magnetic separator
3.7. Place the tube in the home-made magnetic separator for 2~3 min, recover 35 µl of supernatant and subject it for sequencing.