TITLE: Serapure Preparation and Testing
AUTHORS: rute.carvalho Carvalho

[DESCRIPTION]
This protocol is used to prepare low-cost SPRI beads used for Illumina Library preparations. As part of the Hakai Institute Ocean Observing Program, from 0 m to near bottom (260 m), biomolecular samples have been collected weeklyto genetically characterize plankton communities in the Northern Salish Sea since 2015. These SPRI beads have been used to clean up PCR products of 16S, 18S, COI, and 12S amplicons, and implemented as part of a standard procedure for eDNA analysis.

This protocol is a modification from the following publication:
 

Modified by B. Faircloth & T. Glenn November 19, 2011
Ecol. and Evol. Biology, UCLA

[STEPS]
SECTION: QUALITY CONTROL
43. The following image compares the results of “purifying” a mix of 2 µL Fermentas Ultra Low
Range Ladder + 18 µL dH20 using several different amounts of AMPure or Serapure solution to
DNA solution. AMPure is on the left, “Serapure” is on the right. After preparing 20 µL of ladder
+ water mix, we combined that with the volumes of AMPure or Serapure listed below and then
purified using the standard protocol:

 

As you can see, the volume of AMPure or SeraPure controls the size of fragments recovered.
More specifically, the ratio of PEG solution used to the volume of the DNA in the solution 
makes the difference, not the count of beads in the solution (provided they are above the minimum
level). This is what makes it possible to do “double-SPRI” size selection.
SECTION: STEPS
2. In a 50 mL conical using sterile stock solutions, prepare TE (10 mM Tris-HCl, 1 mM EDTA) by adding:
 500 µL    
100 µL    
Fill conical to 50 mL mark with dH20.

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3. Mix the container of    and transfer 1 mL to a 1.5 mL microtube.

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4. Place SpeedBeads on amagnet stand until beads are drawn to magnet.
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5. Remove supernatant with P200 or P1000 pipetter.
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6. Add 1 mL TE to beads, remove from the magnet, mix, and return to the magnet.

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7. Remove supernatant with P200 or P1000 pipetter.
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10. Add 1 mL TE to beads and remove from magnet. Fully resuspend and set microtube in the rack (i.e. not on magnet stand).

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11. Add 9 g   to a new 50 mL, sterile conical.

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12. Add 10 mL    (or 2.92 g NaCl) to conical.

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13. Add    to conical.

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14. Add 100 uL    to conical.

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15. Fill conical to ~ 49 mL using sterile dH20. You can do this by eye, just go slowly.
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16. Mix conical for about 3-5 minutes until PEG goes into solution (solution, upon sitting, should be clear).
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17. Add 27.5  µL    to conical and mix gently.

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18. Mix the 1mL SpeedBead + TE solution and transfer to 50 mL conical.

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19. Fill conical to 50 mL mark with dH20 (if not already there) and gently mix 50 mL conical until brown.
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20. Test against AMPure XP using aliquots of ladder (Fermentas GeneRuler). I recommend the 50 bp ladder in place of the ultraNlow range ladder.
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21. Wrap in tinfoil (or place in dark container) and store at 4°C.

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23. Test monthly.
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22. 
 

SECTION: TESTING
25. Prep fresh aliquots of 70% EtOH.

SECTION: TESTING
26. Mix 2 µL GeneRuler with 18 µL dH20.

SECTION: TESTING
27. Add 20 µL GeneRuler mixture to a volume of Serapure and/or AMPure (the specific volume depends on whether you are trying exclude small fragments or not; see the figure on the next page).

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28. Incubate mixture for 5 min at room temperature.

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29. Place on magnet stand.
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30. Remove supernatant.
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31. Add 500 µL 70% EtOH.

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32. Incubate on stand for 1 min.

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33. Remove supernatant.
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34. Add  500 µL 70% EtOH.

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35. Incubate on stand for 1 min.

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36. Remove supernatant.
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37. Place beads on 37°C heat block for 3-4 min. until dry.

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38. Rehydrate with 20 µL dH20.

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39. Place on magnet stand.
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40. Transfer the supernatant to a new tube. 
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41. Mix supernatant with 1 µL loading dye.

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42. Electrophorese in 1.5 % agarose for 1h at 100 V.

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24. 
 

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9. Remove supernatant with P200 or P1000 pipetter.
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8. Add 1 mL TE to beads, remove from the magnet, mix, and return to the magnet.

SECTION: PREPARATIONS
1. Materials: