TITLE: Serapure Preparation and Testing
AUTHORS: Andreas Novotny

[DESCRIPTION]
A protocol template created through the BeBOP project for DNA Extraction.

[BEFORE_START]
Read background information, MIOP and BePOP-OBON information under the "Guidelines" tab.

[GUIDELINES]
 MIOP: Minimum Information about an Omics Protocol
 
 
AUTHORS
 
This protocol is derived from the referenced protocol created by Nadin Rohland. Please do not
cite this protocol as being originally produced by me.

RELATED PROTOCOLS
 
 Suggested Reading
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
DeAngeliss MM, Wang DG, Hawkins TL: Solid-phase reversible immobilization for the isolation
of PCR products. Nucleic Acids Res 1995, 23:4742–4743.
Fisher S, et al.: A scalable, fully automated process for construction of sequence-ready human
exome targeted capture libraries. Genome Biol 2011, 12:R1.
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.

ACRONYMS AND ABBREVIATIONS
 
 
GLOSSARY
 
 

BACKGROUND

This document describes the required protocol to conduct insert name of the method/protocol.

Summary
The goal here is to create a substitute for AMPure XP that is of equal effectiveness in 
comparison to the commercial product but far more cost-effective ($19/mL versus $0.46/mL).

Insert a short description of the background for the method/protocol (e.g. why and for which purpose do you perform water sampling).
Please provide a brief summary of your method including, as appropriate, a brief description of what techniques your best practice is about, which ocean environments or regions it targets, the primary sensors covered, what type of data/measurements/observing platform it covers, limits to its applicability.
Method description and rationale

Insert a short description of the functioning principal of the methodology used in the protocol (i.e. how does the method work?). Please note that this is different from the step-by-step description of the protocol procedure.
Insert a short statement explaining why the specific methodology used in the protocol has been selected (e.g. it is highly reproducible, highly accurate, procedures are easy to execute etc….).

Spatial coverage and environment(s) of relevance

If applicable, please specify the region where the protocol is applied. For regional term guidance see here. If applicable, please indicate here the environment(s) of relevance for the protocol, e.g. Abyssal plain. Select from the ENVO terminology.

Personnel Required
Insert the number of technicians, data managers, and scientists required for the good execution of the procedure

Safety

Identify hazards associated with the procedure and specify protective equipment and safety training required to safely execute the procedure

Training requirements

Specify technical training required for the good execution of the procedure.

Time needed to execute the procedure

Specify how much time is necessary to execute the procedure.


[STEPS]
SECTION: STANDARD OPERATING PROCEDURE
1. In the following SOP, please use the exact names of equipment as noted in the table above.

Provide a step-by-step description of the protocol. The identification of difficult steps in the protocol and the provision of recommendations for the execution of those steps are encouraged.
SECTION: APPENDIX A: DATASHEETS
45. Link templates (e.g. preformatted spreadsheets) used to record measurements and report on the quality of the data as well as any documents such as manufacturer specifications, images, etc that support this protocol. Please include a short note describing the document’s relevance.
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, it is the ratio of PEG solution used to the volume of the DNA in solution which
makes the difference, not the count of beads in solution (provided they are above the minimum
level). This is what makes it possible to do “doubleNSPRI” size selection.
SECTION: BASIC TROUBLESHOOTING GUIDE
44. Identify known issues associated with the procedure, if any.

Provide troubleshooting guidelines when available.
SECTION: STEPS
2. In a 50 mL conical using sterile stock solutions, prepare TE (10 millimolar (mM) TrisNHCl, 1 millimolar (mM) EDTA = 500 µL 1 Molarity (M) Tris pH8 + 100 µL 0.5 Molarity (M) EDTA, fill conical to 50 mL mark with dH20).

SECTION: STEPS
3. Mix SeraNmag SpeedBeads and transfer 1 mL to a 1.5 mL microtube.

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

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

SECTION: STEPS
11. Add 9 g PEGN8000 to a new 50 mL, sterile conical.

SECTION: STEPS
12. Add 10 mL 5 Molarity (M) NaCL (or 2.92 g) to conical.

SECTION: STEPS
13. Add 500 µL 1 Molarity (M) TrisNHCL to conical.

SECTION: STEPS
14. Add 100 µL 0.5 Molarity (M) EDTA to conical.

SECTION: STEPS
15. Fill conical to ~ 49 mL using sterile dH20. You can do this by eye, just go slowly.
SECTION: STEPS
16. Mix conical for about 3N5 minutes until PEG goes into solution (solution, upon sitting, should be clear).
SECTION: STEPS
17. Add 27.5 µL Tween 20 to conical and mix gently.

SECTION: STEPS
18. Mix 1 mL SpeedBead + TE solution and transfer to 50 mL conical.

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

SECTION: STEPS
23. Test monthly.
SECTION: STEPS
22. 
 

SECTION: TESTING
25. Prep fresh aliquots of 70 Mass / % volume 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).

SECTION: TESTING
28. Incubate mixture 5 min at Room temperature.

SECTION: TESTING
29. Place on magnet stand.
SECTION: TESTING
30. Remove supernatant.
SECTION: TESTING
31. Add 500 µL 70 Mass / % volume EtOH.

SECTION: TESTING
32.   Incubate on stand for 1 min.

SECTION: TESTING
33. Remove supernatant.
SECTION: TESTING
34. Add 500 µL 70 Mass / % volume EtOH.

SECTION: TESTING
35. Incubate on stand for 1 min.

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

SECTION: TESTING
38. Rehydrate with 20 µL dH20.

SECTION: TESTING
39. Place on magnet stand.
SECTION: TESTING
40. Transfer supernatant to new tube. 
SECTION: TESTING
41. Mix supernatant with 1 µL loading dye.

SECTION: TESTING
42. Electrophorese in 1.5 % agarose for 60 min at 100 V.

SECTION: TESTING
24. 
 

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