SPRIselect Bead Protocol for Size Selection

• 85% Ethanol, non-denatured
• Water (molecular biology grade) or a standard buffer solution such as Tris (10 mM, pH 8.0) or TE (10 mM Tris, pH 8.1 mM EDTA) for DNA elution
• Magnet stand or plate

• Samples should be fragmented double-stranded DNA.
• Samples should be dissolved in molecular biology grade water or standard buffer solution such as Tris or TE.
• Sample volume should be ≥ 50 μL. A lower volume will decrease pipetting accuracy of the SPRIselect reagent, therefore increasing selection point variability.
• DNA fragments may be size selected in a range no smaller than 150 bp and no larger than 800 bp.
• To maximize recovery for a left side size selection, the majority of the sample’s size distribution should be larger than the selection point.
• To maximize recovery for a right side size selection, the majority of the sample’s size distribution should be smaller than the selection point.

As a general rule, increasing the ratio of the SPRIselect reagent volume to the sample volume will decrease the efficiency of binding larger fragments. Figure 2 illustrates this relationship.

Figure 2. Agilent High Sensitivity DNA Chip Electropherogram. M = upper and lower markers for High Sensitivity DNA chip. Shear = 1 μL of 20 ng/μL input control sample in water. 1.2x to 0.4x = 1 μL of shear, size selected with given ratio of the SPRIselect reagent volume to the sample volume.

1. Thoroughly shake the SPRIselect bottle to resuspend the SPRI beads. Following the trend depicted in Figure 2, add the required volume of the SPRIselect reagent for the desired ratio to the sample.

   TIP: Volume of sample × ratio = volume of the SPRIselect reagent.
   Example: 50 μL sample × 0.7x ratio = 35 μL of the SPRIselect reagent.

2. Mix the total reaction volume by pipetting 10 times and incubate at RT for 1 minute
   OR
   vortex for 1 minute at an appropriate speed until homogenous (depending on labware and total volume).

   NOTE: Insufficient mixing of the sample and the SPRIselect reagent will lead to inconsistent size selection results. Make sure to mix well.

3. Place the reaction vessel on an appropriate magnetic stand or plate and allow the SPRI beads to settle to the magnet. Settle times will vary – a higher initial sample volume, a higher SPRIselect ratio or weaker magnets will require a longer settle time.

4. Transfer the clear supernatant, which contains the Right Side Size Selected sample, to a new reaction vessel. The reaction vessel with the remaining beads can be discarded.

   NOTE: Care should be taken not to aspirate more than a trace amount of beads during this step, as the undesired larger fragment sizes are associated with the beads. Significant bead transfer will cause tailing into the larger size range.

5. Add the required volume of the SPRIselect reagent, using the calculation below, to the supernatant from step 4 above. This will bind the fragments in the supernatant to the new SPRI beads.

   TIP: Volume of sample μL × (1.8x – the initial ratio) = volume of SPRIselect reagent.
   Example: 50 μL × (1.8x – 0.7x) = 55 μL of SPRIselect reagent.

6. Perform the following:

   • Mix the total reaction volume by pipetting 10 times and incubate at RT for 1 minute
     OR
     vortex for 1 minute at an appropriate speed until homogenous (depending on labware and total volume).

     NOTE: Insufficient mixing of the sample and the SPRIselect reagent will lead to inconsistent size selection results.

   • Place the reaction vessel on an appropriate magnetic stand or plate and allow the SPRI beads to settle to the magnet. Settle times will vary – a higher initial sample volume, a higher SPRIselect ratio or weaker magnets will require a longer settle time.

   • Remove and discard the clear supernatant.

     NOTE: Care should be taken not to aspirate more than a trace amount of beads during this step, as the desired library is associated with the beads. Significant bead loss will result in reduced yield.

7. With the reaction vessel still on the magnet, add 180 μL of 85% ethanol (non-denatured) and incubate at RT for 30 seconds. Remove and discard the ethanol supernatant.

   NOTE: Care should be taken not to aspirate more than a trace amount of beads during this step, as the desired library is associated with the beads. Significant bead loss will result in reduced yield.

8. To elute the sample:

   • Remove the reaction vessel from the magnet and add ≥ 20 μL of molecular biology grade water or standard buffer solution such as Tris or TE.

     NOTE: Elution volume should be large enough so that the liquid level is high enough for the beads to settle to the magnet.

   • Mix the total elution volume by pipetting 10 times to resuspend the beads and incubate at RT for 1 minute
     OR
     vortex for 1 minute at an appropriate speed until homogenous (depending on labware and total volume).

   • Place the reaction vessel on an appropriate magnetic stand or plate and allow the SPRI beads to settle to the magnet. Settle times will vary; a higher elution volume or weaker magnets will require a longer settle time.

9. Transfer the eluate (size selected sample) to an appropriate storage vessel.