meth_3.1.9 Qiagen DNeasy PowerWater Sterivex Kit modified IMOS NRS ACMON and voyages CSIRO Hobart Genetics Laboratories

Contributor(s): CSIRO Environmental Genomics Team

Citation: Appleyard, S., Abell, G. & Watson, R. Tackling microbial related issues in cultured shellfish via integrated molecular and water chemistry approaches. Seafood CRC report No. 2011/729 (2011).

AM Project ID: AM_IMOS, AM0001

Reagents:

• Lysis buffer
  • 200 mM NaH₂PO₄2H₂O (monobasic)
  • 200 mM Na₂HPO₄ (dibasic)
  • To make up 200 mL lysis buffer:
    • 39 mL 200 mM NaH₂PO₄
    • 61 mL 200mM Na₂HPO₄
    • 17.54 g NaCl
    • 2 g CTAB
    • 4 g PVP K30
    • + ddH₂0 to make up to 200 mL
    • Adjust to pH 7.0 (using NaOH – try couple of mL of 10 M NaOH)
  • Lysozyme
  • Proteinase K – 20 mg/mL
  • MT Buffer (MP Biomedicals)
  • From Qiagen DNeasy® PowerWater® Sterivex™ Kit (https://www.qiagen.com/au/products/discovery-and-translational-research/dna-rna-purification/dna-purification/microbial-dna/dneasy-powerwater-sterivex-kit/?clear=true#orderinginformation)
    • Columns and sample recovery tubes, 3 mL and 20 mL syringes, 5 mL tubes Buffer
    • MR Buffer (warmed to 65 °C before use)
    • Ethanol and PW
  • Inlet and outlet caps for Sterivex filters
  • Phenol:Chloroform:Isoamyl (25:24:1) (PCI) Chloroform:Isoamyl (24:1) (CI)
  • TE buffer

Method:

(Processing limited by capacity of horizontal vortexer, 2 vortexers x 6 filters = 12 filters per extraction set)

1. weigh 125 mg lysozyme into 50 mL falcon tube and add 25 mL Lysis Buffer to dissolve (lysozyme final conc. 5 mg/mL).
2. remove filters from -80 °C, remove inlet cap and using a pipette add 1.875 mL Lysis buffer and 125 µL MT buffer. (if filters are covered with RNAlater, remove RNAlater with back pressure from 3 mL syringe)
3. recap the Sterivex filter and attach filter (with inlet end facing out) to the horizontal vortexer, Speed 5-7 for 60 min (turning the filter a couple of times during the h)
4. using 3 mL syringe, draw back plunger and attach to inlet end of filter until pressure builds up – release plunger and buffer in filter should flow into syringe. Divide approx 2 mL of buffer evenly into 2 × 2.0 mL tubes (do not use the 2 mL collection tubes that come in the PowerWater® Sterivex kit – they don’t tolerate PCI) (may need to use syringe several times to get all buffer out of filter, should be about 0.800-1.00 mL per tube)
5. in fume hood, add 900 µL PCI to each tube, invert several times, spin down 13000/10 min/RT
6. combine the aqueous phases from both tubes into one 2.0 mL tube (which will be between 1.2 – 1.5 mL), add 20 µL Proteinase K, onto heat block for 2 h at 60 °C
7. in fume hood, add 500 μL CI, spin down 13000/10 min/RT – put aqueous phase into new tube
8. in fume hood, add a further 500 μL CI, spin down 13000/5 min/RT – put aqueous phase into new tube
9. after 2^nd spin, take out 1 mL of aqueous phase, add to 5 mL tube
10. add 3 mL of warmed MR buffer (65 °C), mix by inversion
11. attach column to barrel of 20 mL syringe and attach to vacuum manifold
12. pour contents of 5 mL tube into barrel while still warm
13. using vacuum, pull contents through the column
14. while keeping column attached to the manifold, remove barrel and add 800 μL Ethanol to column
15. using vacuum, pull contents through the column
16. add 800 µL PW to column
17. using vacuum, pull contents through the column, then keep on vacuum for 2 mins
18. turn vacuum off, put column into new 2.0 mL tube and let air dry on bench for 10 mins
19. add 80 µL 0.1 x TE to column, incubate at 37 °C for 45 min
20. spin down column and tube at 13000/2 min/RT to elute DNA