TITLE: Preparation of Sera-Mag SpeedBeads
AUTHORS: Nina Alperovich

[DESCRIPTION]
This protocol describes the preparation of Sera-mag SpeedBeads with PEG-salt buffer for downstream use in the Automated Bar-Seq Library Preparation protocol.

[STEPS]
SECTION: Prepare SpeedBeeds in tubes
1. Vortex 15 mL Sera-mag SpeedBeads solution until beads are uniformly distributed. 
SECTION: Prepare SpeedBeeds in tubes
3. Place all 15 of the tubes on the tube magnetic separation rack until beads are drawn to magnet.
SECTION: Prepare SpeedBeeds in tubes
4. Remove the supernatant from each tube.
SECTION: Prepare SpeedBeeds in tubes
2. Transfer 1 mL to each of the fifteen 2 mL microcentrifuge tubes.
SECTION: Perform the first wash step
5. Add 1 mL TE buffer to each tube.
SECTION: Perform the first wash step
6. Remove the tubes from the magnetic stand and mix by vortexing
SECTION: Perform the first wash step
7. Return tubes to the magnetic separation rack and wait until beads are drawn to magnet.
SECTION: Perform the first wash step
8. Remove supernatant from each tube.
SECTION: Perform the second wash step
9. Add 1 mL TE buffer to each tube.
SECTION: Perform the second wash step
10. Remove the tubes from the magnetic stand and mix by vortexing
SECTION: Perform the second wash step
11. Return tubes to the magnetic separation rack and wait until beads are drawn to magnet.
SECTION: Perform the second wash step
12. Remove supernatant from each tube.
SECTION: Resuspend the beads
13. Add 1 mL TE to each tube and remove from magnet. 
SECTION: Resuspend the beads
14. Fully resuspend by vortexing and set in a non-magnetic tube rack (not on magnetic stand).
Note: Beads that have been washed and resuspended in TE can be stored in the refrigerator at this point and used for the following steps as needed.
SECTION: SpeedBeads magnetic beads/PEG/NaCl buffer preparation
15. Add 9 g PEG-8000 to a 50 mL sterile conical tube.
SECTION: SpeedBeads magnetic beads/PEG/NaCl buffer preparation
16. Add 10 mL 5 mol/L NaCl to the conical tube.
SECTION: SpeedBeads magnetic beads/PEG/NaCl buffer preparation
17. Add 500 µL 1 mol/L Tris-HCl, pH8 to the conical tube.
SECTION: SpeedBeads magnetic beads/PEG/NaCl buffer preparation
18. Add 500 µL 0.5 mol/L EDTA to the conical tube.
SECTION: SpeedBeads magnetic beads/PEG/NaCl buffer preparation
19. Fill the conical tube to ~45 mL using sterile nuclease free water.
SECTION: SpeedBeads magnetic beads/PEG/NaCl buffer preparation
20. Mix the conical tube by vortexing every 1-2 minutes until PEG goes into solution.  
When not vortexing, the tube can be placed on a heated shaker (e.g., Eppendorf ThermoMixer) at 50°C, 600 rpm, to reduce time required to dissolve the PEG
 When finished, the solution should be clear.
SECTION: SpeedBeads magnetic beads/PEG/NaCl buffer preparation
21. Add 27.5 μL Tween 20 to the conical tube and mix gently by inverting 4-6 times.
SECTION: SpeedBeads magnetic beads/PEG/NaCl buffer preparation
22. Mix two 1 mL aliquots of the SpeedBeads + TE solution until the solution is a uniform brown color, then transfer the solutions from both tubes to the 50 mL conical tube.
SECTION: SpeedBeads magnetic beads/PEG/NaCl buffer preparation
23. Fill the conical tube to the 50 mL mark with sterile nuclease free water
SECTION: SpeedBeads magnetic beads/PEG/NaCl buffer preparation
25. Wrap in the conical tube in foil (or place in dark container) and store at 4 °C until use.
SECTION: SpeedBeads magnetic beads/PEG/NaCl buffer preparation
24.  Gently mix until the solution is a uniformed brown color.