TITLE: DNA Extraction from Sterivex Filters - Qiagen Blood and Tissue Kit
AUTHORS: Colleen Kellogg

[DESCRIPTION]
Draft!

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

[GUIDELINES]
 MIOP: Minimum Information about an Omics Protocol
 
 
AUTHORS
 

RELATED PROTOCOLS
 
 
ACRONYMS AND ABBREVIATIONS
 
 
GLOSSARY
 
 

BACKGROUND

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

Summary

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: PREPARATION - must be done in Clean Room 1 or Clean Room 2
1. 
 
  

SECTION: PREPARATION - must be done in Clean Room 1 or Clean Room 2
4. Set aside ATL buffer, AL buffer, proteinase K, RNase A and anhydrous ethanol. 
Put the ATL and AL buffers in the incubator to eliminate any precipitate that may be in the solution. 
There is a cardboard box in the mini freezer with pro K aliquots. Each aliquot has enough volume for 12 samples. The anhydrous ethanol is stored in the CR1 mini fridge, and in the CR2 in the mini freezer.
SECTION: PREPARATION - must be done in Clean Room 1 or Clean Room 2
5. Calculate the volume of each reagent you will need for each step and have a one-time-use tube to make an aliquot for that specific reagent. Try to add a little bit more than you need and dispose of the left over. Prepare the aliquots inside the workstation (with HEPA filter).
SECTION: PREPARATION - must be done in Clean Room 1 or Clean Room 2
6. Cut the parafilm to a size of 1 cm x 5 cm, two or three per Sterivex – sometimes you will need to replace the parafilm in the Sterivex.
SECTION: PREPARATION - must be done in Clean Room 1 or Clean Room 2
3. Wipe down the benches, centrifuge, and working areas using the PREempt solution/wipes. 
Turn on the incubator and set the temperature to 56 °C.
SECTION: BEADS BEATING AND INITIAL INCUBATION
7. Thaw the Sterivex filters.
SECTION: BEADS BEATING AND INITIAL INCUBATION
8. Remove the parafilm and remove Longmire’s buffer/SLB using a syringe. You can use a plastic “tripour” to dispose the buffer and then pour all of the volume in the sink. Rinse the tripour with water after use, dry and wipe it down using a paper towel and PREempt solution.
SECTION: BEADS BEATING AND INITIAL INCUBATION
9. If needed, dry the Sterivex inlet using a Kimwipes (one wipe per Sterivex). Place the Sterivex on a clean Kimwipe while preparing the other one Sterivexes.
SECTION: BEADS BEATING AND INITIAL INCUBATION
10. Add 0.1 mm silica beads (about 0.3 g or three spoons*) into the Sterivex using a weighting paper (make a funnel with the paper to slide in the beads, use one per Sterivex). *It is a white spoon that is stored in the drawer with the silica beads.
SECTION: BEADS BEATING AND INITIAL INCUBATION
11. Seal the outlet port of the Sterivex filter unit with the parafilm.
SECTION: BEADS BEATING AND INITIAL INCUBATION
12. Inject 720 µL ATL buffer into the Sterivex.

SECTION: BEADS BEATING AND INITIAL INCUBATION
13. One more Sterivex +ATL buffer should be prepared for the extraction blank for detecting contamination during DNA extraction.
SECTION: BEADS BEATING AND INITIAL INCUBATION
14. Place the Sterivex in the vortex adapter and do the “bead-beating” for 10 min to promote cells lysis.
SECTION: BEADS BEATING AND INITIAL INCUBATION
15. Get one 50mL tube per sample, and put the Sterivexes inside the 50mL tube. 
Incubate the Sterivex at 56 °C for 30 min using the rotisserie.
SECTION: BEADS BEATING AND INITIAL INCUBATION
16. Repeat the steps 8 and 9 once again.
SECTION: DIGESTION WITH PRO-K AND INCUBATION
17. Add 80 µL proteinase-K to each Sterivex.

SECTION: DIGESTION WITH PRO-K AND INCUBATION
18. Seal the outlet port of the Sterivex filter unit with a parafilm if you notice that some lysate is leaking.
SECTION: DIGESTION WITH PRO-K AND INCUBATION
19. Vortex tubes for ~10 s (or simply move it to mix the solution). 
Place the Sterivexes back to the 50mL.
Incubate tubes at 56 °C for 120 min using the rotisserie.

SECTION: COLLECTING THE LYSATE
20. While warming the Sterivex filter unit to 56 °C, prepare a 2.0 mL tube for DNA recovery (loBind DNA tubes). Note: write the necessary information on the cap of the 2.0 mL tube.

SECTION: COLLECTING THE LYSATE
21. After completion of warming, carefully remove the parafilm and the luer fitting on the inlet port of the Sterivex filter unit, while preventing liquid inside from leaking.
SECTION: COLLECTING THE LYSATE
22. Insert the inlet port of the Sterivex filter unit into the 2.0 mL tube and lightly push it down to the bottom of the 50mL conical tube. Then, close the cap of the conical tube firmly.

SECTION: COLLECTING THE LYSATE
23. Put the conical tubes in the Megafuge adapters. 
Centrifuge the conical tube containing the Sterivex filter unit at 6,000 x g for 1 min and collect the extracted DNA in a 2 mL tube.

SECTION: COLLECTING THE LYSATE
24. Remove the 50 mL conical tube from the centrifuge and remove the Sterivex filter unit and 2.0 mL tube using tweezers.
Note: The 2.0 mL tube is uncapped; handle it carefully. Wipe down the tweezers with PREempt wipes between samples.
SECTION: COLLECTING THE LYSATE
25. Discard the used conical tubes (keep one conical tube and use it as a provisory waste container) and Sterivex filter unit. 
Firmly cap the 2.0 mL tube and proceed with these vials and lysate
SECTION: FINAL INCUBATION AND PRECIPITATION
26. Add 4 µL of RNAse A (100 mg/mL). 
Mix pipetting up and down (or vortexing). 
Incubate at Room temperature for 2 min.

SECTION: FINAL INCUBATION AND PRECIPITATION
27. Add 200 µL buffer AL. 
Mix thoroughly pipetting up and down (or by vortexing). It may form some precipitate. 
Incubate at 56 °C for 10 min (it doesn’t need to be in the rotisserie).

SECTION: FINAL INCUBATION AND PRECIPITATION
28. Add 200 µL ethanol (96-100%). 
Mix thoroughly pipetting up and down (or by vortexing). 
Total volume now is ~1.2 mL.
SECTION: DNA BINIDNG AND WASHING
29. Pipette 600 µL the mixture into a DNeasy mini spin column placed in a 2 mL collection tube. 
Place the tubes in the centrifuge and spin the tubes at 8,000 rpm (~6,000 x g) for 1 min. 
Discard the flow through.
SECTION: DNA BINIDNG AND WASHING
30. Repeat the step 22 once again using the remaining volume of the mixture from step 21.
SECTION: DNA BINIDNG AND WASHING
31. Place the spin column in a new 2 mL collection tube. If after spinning down you still can see some buffer in the spin column, centrifuge the tube again with a higher speed (about 10,000 x g). If this still not work, spin down with a higher speed.
Add 500 µL buffer AW1. 
Centrifuge for 1 min at 8,000 rpm (~6,000 x g). 
Discard the flow through and collection tube.
SECTION: DNA BINIDNG AND WASHING
32. Place the spin column in a new 2 mL collection tube. Add 500 µL buffer AW2. 
Centrifuge for 3 min at 14,000 rpm (~20,000 x g). 
Discard the flow through and collection tube.
SECTION: DNA BINIDNG AND WASHING
33. Transfer the spin column to a new 1.5 mL or 2 mL microcentrifuge tube.
SECTION: DNA ELUTION
34. Elute the DNA by adding 200 µL buffer AE to the center of the spin column membrane. 
Incubate for 1 min at 15-20 Room temperature. 
Centrifuge for 1 min at 8,000 rpm (~6,000 x g).

SECTION: DNA ELUTION
35. OPTIONAL: repeat the step 27 for increased DNA yield.
SECTION: PREPARATION - must be done in Clean Room 1 or Clean Room 2
2. UV for 30 minutes the following:  
Silica beads - you will need a 5mL tube full of beads to extract DNA from 12 samples 
(See bench top guide in the end of this protocol to check the amount of tubes you will need)
2mL tubes
50mL falcon tubes
Racks
Pipettes 
pipette tips

Do not forget to UV tubes for that will be used to aliquot the buffers, and silica beads. 
36.