TITLE: SPRI Bead Cleanup
AUTHORS: George  Testo

[DESCRIPTION]
SPRIselect is a SPRI-based chemistry that speeds and simplifies nucleic acid size selection for fragment library preparation for Next Generation sequencing. In this process, size selection is required to produce a uniform distribution of fragments around an average size. Using SPRIselect, the size distribution can be adjusted to suit the application and platform used. The process can be scaled for low to high throughput workflows.

The guidance provided below can be used to optimize the desired size selection range. Used manually or automated on a liquid handling system such as the Biomek Laboratory Automation Workstation, SPRIselect will provide rapid and consistent size selection suitable for most applications.

[BEFORE_START]
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. See Effects of Common Laboratory Reagents on Size Selection at end of user guide for more information.

Sample volume should be ≥ 50 μL. A lower volume will decrease pipetting accuracy of SPRIselect, 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.

To maximize recovery for a Double Size Selection, the size distribution should be centered between the selection points.

[STEPS]
SECTION: Preparing for Bead Cleanup
1. Make 300 µL 80% Ethanol (200-proof) (make fresh each time). Gently shake the SPRI beads bottle to resuspend any magnetic particles that may have settled. Take aliquot into a tube for working solution.

SECTION: Preparing for Bead Cleanup
2. Pipette PCR product (single or pooled) and transfer to single 0.2mL tube(s).
SECTION: Preparing for Bead Cleanup
3. Add 1.2X the volume of SPRI beads to the samples.
SECTION: Preparing for Bead Cleanup
3.1. Total volume of your sample or pool x 1.2 = required volume of SPRI beads
SECTION: Preparing for Bead Cleanup
4. Mix reagent and PCR product thoroughly by pipette mixing.
SECTION: Performing Bead Cleanup
5. Incubate mixed samples for 10 min at room temperature for maximum recovery. Making sure to tap gently every 2-3 minutes.

 


SECTION: Performing Bead Cleanup
6. Place the tube onto the magnetic stand for 2 min to separate beads from the solution.

 

SECTION: Performing Bead Cleanup
7. Remove supernatant and discard.

 

SECTION: Performing Bead Cleanup
8. Add 120 µL of 80% Ethanol to the 1.5mL tube on the stand and incubate for 1 min at room temperature. Pipette off the ethanol and discard. Repeat for a total 2 washes.

 

SECTION: Performing Bead Cleanup
9. Open the tube lid(s) to allow evaporation of excess ethanol 5 min.

 

SECTION: Retrieving Desired PCR Product
10. Remove the tube(s) from the stand. Add 40 µL of elution buffer (from the extraction kit) and mix by pipetting. This will separate the beads from the PCR product.

 


SECTION: Retrieving Desired PCR Product
11. Place the tube(s) back onto the magnetic stand for 1 min to separate the beads from the solution. Transfer the eluent to a new tube. This contains the purified PCR product.