TITLE: [Modified] Lake ABPS Protocol - University of Maine
AUTHORS: Grayson Huston

[DESCRIPTION]
A modified version of the Lake ABPS protocol as described in Thomson-Laing et al. 2022

Protocol successful at detecting fish sedDNA collected from lake surface sediments, as well as river sediments during an anadromous fish sea-run migration

[STEPS]
SECTION: Alkaline Lysis &amp; Ethanol Precipitation
1. CENTRIFUGE sediment samples at 5250 x g  for 5 min 

DISCARD pore water using a sterile pipette, so only sediment remains
SECTION: Alkaline Lysis &amp; Ethanol Precipitation
2. ADD 10 g of wet sediment to a sterile 50 mL tube

ADD 6 mL sodium hydroxide (NaOH, 0.33M) to the sample

ADD 3 mL Tris-EDTA (pH 8.0) to the sample                                                                                                                                                                                                                                                   
SECTION: Alkaline Lysis &amp; Ethanol Precipitation
4. CENTRIFUGE samples at 5250 x g for 60 min 

TRANSFER 7.5 mL of supernatant to a new, sterile 50 mL tube
SECTION: Alkaline Lysis &amp; Ethanol Precipitation
5. ADD 7.5 mL Tris-HCl (pH 6.7)

ADD 1.5 mL sodium acetate (3M, pH 5.2)

ADD 30 mL molecular grade ethanol
SECTION: Alkaline Lysis &amp; Ethanol Precipitation
6. INCUBATE samples at -20 °C  for 60 min
SECTION: Alkaline Lysis &amp; Ethanol Precipitation
7. CENTRIFUGE sample at 5250 x g  for 60 min 

DISCARD supernatant

ALLOW remaining ethanol to evaporate off of concentrated pellet before proceeding to next step
SECTION: PowerSoil Pro Extraction on Concentrated Pellet - sample preparation &amp; cell lysis
8. WEIGH the concentrated pellet and split it into multiple 0.5 g replicates

TRANSFER each 0.5 g replicate into a PowerBead Pro Tube
SECTION: PowerSoil Pro Extraction on Concentrated Pellet - sample preparation &amp; cell lysis
9. ADD 800 µL of Solution CD1 to each PowerBead Pro Tube

SECURE PowerBead Pro Tubes horizontally to a Vortex Adapter

VORTEX for 10 min 

ROTATE tubes so caps are oriented in the opposite direction

VORTEX for another 10 min 
SECTION: PowerSoil Pro Extraction on Concentrated Pellet - sample preparation &amp; cell lysis
10. CENTRIFUGE sample at 15000 x g  for 2 min 

TRANSFER all supernatant to a clean 2 mL Microcentrifuge Tube
SECTION: PowerSoil Pro Extraction on Concentrated Pellet - inhibitor removal
11. ADD 200 µL of Solution CD2 

VORTEX briefly to mix
SECTION: PowerSoil Pro Extraction on Concentrated Pellet - inhibitor removal
12. CENTRIFUGE at 15000 x g for 1 min 

AVOIDING the pellet, transfer all supernatant to a clean 2 mL Microcentrifuge Tube
SECTION: PowerSoil Pro Extraction on Concentrated Pellet - bind DNA
13. ADD 600 µL of Solution CD3

VORTEX briefly to mix
SECTION: PowerSoil Pro Extraction on Concentrated Pellet - bind DNA
14. LOAD 650 µL of lysate onto an MB spin column

CENTRIFUGE at 15000 x g for 1 min 

DISCARD the liquid flow-through
SECTION: PowerSoil Pro Extraction on Concentrated Pellet - bind DNA
15. REPEAT step 14 to ensure all the lysate has passed through the MB Spin Column

CAREFULLY place the MB spin column into a clean 2mL collection tube
SECTION: PowerSoil Pro Extraction on Concentrated Pellet - wash spin column
16. ADD 500 µL of Solution EA to the MB spin column

CENTRIFUGE at 15000 x g for 1 min 

DISCARD the liquid flow-through and place the MB spin column into the same 2 mL Collection Tube
SECTION: PowerSoil Pro Extraction on Concentrated Pellet - wash spin column
17. ADD 500 µL of Solution C5 to the MB spin column

CENTRIFUGE at 15000 x g for 1 min 

DISCARD the liquid flow-through and place the MB Spin Column into a new 2 mL Collection Tube
SECTION: PowerSoil Pro Extraction on Concentrated Pellet - wash spin column
18. CENTRIFUGE at 16000 x g for 2 min

CAREFULLY place the MB spin column into a new 2mL Collection Tube 

SECTION: PowerSoil Pro Extraction on Concentrated Pellet - wash spin column
19. ADD 50-100 µL of Solution C6 to the center of the white membrane in the MB Spin Column
 

SECTION: PowerSoil Pro Extraction on Concentrated Pellet - wash spin column
20. CENTRIFUGE at 15000 x g for 1 min

DISCARD the MB Spin Column

POOL all replicates into a sterile 1.5 mL Microcentrifuge Tube

DNA is now ready for downstream applications
 

SECTION: Alkaline Lysis &amp; Ethanol Precipitation
3. VORTEX sample at max speed for 1 min 

INCUBATE sample at 65 °C for 55 min 

ALLOW samples to cool to Room temperature