TITLE: Home-Brew SPRI Beads
AUTHORS: Sarah Boswell

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The is a protocol to make a substitute for AMPure XP that is of equal effectiveness in comparison to the commercial product but far more cost-effective.</div><div class = "text-block">Credit for this goes to the developers of this protocol:</div><div class = "text-block"><span>Ethan Ford;</span><a href="https://ethanomics.wordpress.com" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://ethanomics.wordpress.com/</span></a></div><div class = "text-block">B. Faircloth & T. Glenn</div><div class = "text-block">November 19, 2011</div><div class = "text-block">Ecol. and Evol. Biology</div><div class = "text-block">Univ. of California – Los Angeles</div><div class = "text-block">This protocol is derived from the referenced protocol created by Nadin Rohland. </div><div class = "text-block"><span style = "font-weight:bold;">Suggested Reading</span></div><div class = "text-block"><a href="http://core-genomics.blogspot.com/2012/04/how-do-spri-beads-work.html" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">http://core-genomics.blogspot.com/2012/04/how-do-spri-beads-work.html</span></a></div><div class = "text-block">Rohland N, Reich D.  Cost-­‐effective, high-­‐throughput DNA sequencing libraries for multiplexed target capture.  Genome Research.  Early Online Access.  Doi: 10.1101/gr.128124.111</div><div class = "text-block">DeAngelis MM, Wang DG, Hawkins TL: Solid-­‐phase reversible immobilization for the isolation of PCR products. Nucleic Acids Res 1995, 23:4742–4743.</div><div class = "text-block">Fisher S, et al.: A scalable, fully automated process for construction of sequence-­‐ready human exome targeted capture libraries. Genome Biol 2011, 12:R1.</div><div class = "text-block">Lundin S, Stranneheim H, Pettersson E, Klevebring D, Lundeberg J: Increased throughput by parallelization of library preparation for massive sequencing. PLoS One 2010, 5:e10029.</div></div>

[STEPS]
?. [Make TE solution (10mM TrisHCl, 1mM EDTA)]
Make TE solution (10mM Tris-HCl, 1mM EDTA pH 8)  1M Tris-HCl pH 8  0.5M EDTA  Nuclease free water
500 µl
100 µl
49.6 mL
?. [Wash SpeedBeads]
Mix SpeedBead bottle thoroughly by shaking and vortexing.
?. [Wash SpeedBeads]
Transfer  of SpeedBeads to a 1.5ml microtube.
1 mL
?. [Wash SpeedBeads]
Place microtube on magnetic stand until the beads are drawn to magnet.
?. [Wash SpeedBeads]
Remove and discard supernatant.
?. [Wash SpeedBeads]
Add  to the beads and remove from the magnet.
[TE]
?. [Wash SpeedBeads]
Remove microtube from magnet and resuspend beads by mixing or vortexing.
?. [Wash SpeedBeads]
This will be a total of 3 washes.Leave washed beads on rack until needed in Step 16.
[Repeat steps 4-7 twice]
?. [Make SPRI Buffer]
Add  directly to a new sterile 50ml conical tube.Do this in chemical room. Tear the balance to 0 with the tube on the balance in a holder. Then weight out 9g into the tube. Seal tube and bring to bench to finish buffer.
[PEG-800]
?. [Make SPRI Buffer]
Add  to the 50ml conical.
[5M NaCl]
?. [Make SPRI Buffer]
Add  to the 50ml conical.
[1M Tris-HCl pH8]
?. [Make SPRI Buffer]
Add  to the 50ml conical.
[0.5M EDTA pH8]
?. [Make SPRI Buffer]
Fill the 50ml conical to ~ 48 mL using nuclease free dH2O. You can do this by eye, just go slowly. I add 2 mL of water to get to about 20 mL then add 28.5 mL of water.
?. [Make SPRI Buffer]
Mix conical for about 5-20 minutes until PEG goes into solution (solution, upon sitting, should be clear). The exact time of mixing is not critical. You can leave it longer without issue.
?. [Make SPRI Buffer]
Add  to the 50ml conical and mix gently.
[10% Tween20]
?. [Mix Beads & Buffer]
Resusped the SpeedBead TE solution from Step 8 then add it to the 50ml conical with the SRPI buffer.
I try to add the beads to the bottom of the 50ml conical.
?. [Mix Beads & Buffer]
Take buffer from the top of the 50ml conical tube and rinse out the eppendorf tube which contained the SpeedBeads to recover all the beads.
?. [Mix Beads & Buffer]
Fill conical to 50ml mark with dH2O (if not already there) and gently mix 50ml conical until a uniform brown color.
?. [Storage]
Wrap conical in tinfoil and store at 4C.
I usually prep 4 x 50ml conicals at one time. Then pool them into an empty nuclease free water bottle for storage.
?. [Validate home-brew SPRI]
Test Home-Brew SPRI agains Ampure XP.  I use total RNA and fragmented RNA to perform a head-to-head. You can also test using a cDNA library. Using RNA as the input serves as validation that there is no RNase activity in the solution.
?. [Validate home-brew SPRI]
Prepare fresh 80% ethanol.
?. [Validate home-brew SPRI]
Purify Total RNA and Fragmented RNA at 1x and 2x SPRI ratio. You can test whatever ratios you need to confirm are working for your assay. These are good tests for my use.Run purified sample on BioAnalyzer.
?. [Validate home-brew SPRI]
Total RNA 2x SPRI purification test results - 100ng/ul input
?. [Validate home-brew SPRI]
Fragmented RNA 1x SPRI purification test results - 15ng/ul input