<commit_before># Manure or manure-fed bioreactor sample DNA extraction
Author: <NAME>    


## Objectives   
Obtain total DNA from manure or manure-fed bioreactor samples for downstream molecular analyses, including PCR and sequencing.    
This procedure includes two parts:    
1. Subsampling     
2. DNA extraction   

## Part 1. Subsampling 

### Materials   
1. 1 ml pipetteman   
2. 1,000 µl or 1,250 µl pipette tips   
3. razor blade    
4. disposable gloves    
5. [MoBio PowerSoil htp-96 well DNA extraction kit](https://mobio.com/products/high-through-put/soil/powersoilr-htp-96-well-soil-dna-isolation-kit.html)    

### Procedures 
1. Preperation (at least 1 day before subsampling):
    1. Clean the bench top using 70% ethanol.      
    1. Prepare wide-bore tips   
        1. wear disposable gloves    
        1. take a non-barrier 1,000 µl or 1,250 µl pipette tip
        2. wipe the razor blade clean using 70% ethanol if it's covered with dust or dirt     
        3. cut the tip using the clean razor blade for an opening ~4 mm         
             + you may be able to buy these, but make sure the opening is wide enough. Else you may not able to pipette the samples through.    
        4. rebox the tips like normal pipette tips. Label it well to indicate it contains wide-bore tips.    
        5. autoclave to sterilize 
    
2. Subsampling (may take multiple days):    
    1. Clean the bench top using 70% enthanol.    
    2. Thaw your samples ahead of time.        
    3. Take a bead plate from MoBio PowerSoil htp-96 well DNA extraction kit.    
    4. Decide the right orientation of the plate (ie., A1 at the left top corner).    
        + label plate number to the side of the plate    
        + label plate number on the silicon lid as well (this will also help you to determine the silicon lid orientation)    
        + the column letters are difficult to see. I color them with marker and it works well.   
    5. Laydown a papertowel on the benchtop then lay a kim-wipe on top of it.    
        + this create a clean spot to lay your bead plate silicon lid    
    6. Carefully lift up the silicon lid from the bead plate and lay it upside down on the kim-wipe you put down in previous step.       
        + the beads are loose. If you yank the silicon lid too hard, beads are going to fly out.    
        + I usually put another kim-wipe on the upside down silicon lid so that nothing falls on it during subsampling.    
    7. Take a thawed sample, write-down the sample label and the well it is going into (e.g., A1) in your notebook.    
    8. With the wide-bore tips you prepared, pipette 250µl manure materials into well A1.   
        + be gentle and careful when pipetting. Even with wide-bore tips, the solids in manure may clog your tips and cause the manure being sucked up into the pipetteman itself.    
        + if the samples got into the pipetteman, take the pipetteman apart to clean before proceding. If not, you will be cross contaminating all of your samples.    
    9. Repeat step 7 and 8 until the entire 96 well bead plate is filled. Store the filled plate at -80ºC until extraction.    
        + because the centrifuge, it works the best if two plates were extracted at the same time.     
    10. If you need multiple days to fill one plate, put the silicon lid back on (and seal it tight) in the exact orientation.    
        + you don't want to introduce cross contamination.    
        + store the plate in -80ºC.   


## Part 2. DNA extraction    

### Materials    
1. Multi-channel pipettemen    
2. Autoclaved pipette tips and barrier pipette tips (various volumes)   
3. Disposable media trays    

### Procedures (mostly folow the kit protocol with some modifications)    <commit_msg>adding extraction procedures<commit_after># Manure or manure-fed bioreactor sample DNA extraction
Author: <NAME>    


## Objectives   
Obtain total DNA from manure or manure-fed bioreactor samples for downstream molecular analyses, including PCR and sequencing.    
This procedure includes two parts:    
1. Subsampling     
2. DNA extraction   

## Part 1. Subsampling 

### Materials   
1. 1 ml pipetteman   
2. 1,000 µl or 1,250 µl pipette tips   
3. razor blade    
4. disposable gloves    
5. [MoBio PowerSoil htp-96 well DNA extraction kit](https://mobio.com/products/high-through-put/soil/powersoilr-htp-96-well-soil-dna-isolation-kit.html)    

### Procedures 
1. Preperation (at least 1 day before subsampling):
    1. Clean the bench top using 70% ethanol.      
    1. Prepare wide-bore tips   
        1. wear disposable gloves    
        1. take a non-barrier 1,000 µl or 1,250 µl pipette tip
        2. wipe the razor blade clean using 70% ethanol if it's covered with dust or dirt     
        3. cut the tip using the clean razor blade for an opening ~4 mm         
             + you may be able to buy these, but make sure the opening is wide enough. Else you may not able to pipette the samples through.    
        4. rebox the tips like normal pipette tips. Label it well to indicate it contains wide-bore tips.    
        5. autoclave to sterilize 
    
2. Subsampling (may take multiple days):    
    1. Clean the bench top using 70% enthanol.    
    2. Thaw your samples ahead of time.        
    3. Take a bead plate from MoBio PowerSoil htp-96 well DNA extraction kit.    
    4. Decide the right orientation of the plate (ie., A1 at the left top corner).    
        + label plate number to the side of the plate    
        + label plate number on the silicon lid as well (this will also help you to determine the silicon lid orientation)    
        + the column letters are difficult to see. I color them with marker and it works well.   
    5. Laydown a papertowel on the benchtop then lay a kim-wipe on top of it.    
        + this create a clean spot to lay your bead plate silicon lid    
    6. Carefully lift up the silicon lid from the bead plate and lay it upside down on the kim-wipe you put down in previous step.       
        + the beads are loose. If you yank the silicon lid too hard, beads are going to fly out.    
        + I usually put another kim-wipe on the upside down silicon lid so that nothing falls on it during subsampling.    
    7. Take a thawed sample, write-down the sample label and the well it is going into (e.g., A1) in your notebook.    
    8. With the wide-bore tips you prepared, pipette 250µl manure materials into well A1.   
        + be gentle and careful when pipetting. Even with wide-bore tips, the solids in manure may clog your tips and cause the manure being sucked up into the pipetteman itself.    
        + if the samples got into the pipetteman, take the pipetteman apart to clean before proceding. If not, you will be cross contaminating all of your samples.    
    9. Repeat step 7 and 8 until the entire 96 well bead plate is filled. Store the filled plate at -80ºC until extraction.    
        + because the centrifuge, it works the best if two plates were extracted at the same time.     
    10. If you need multiple days to fill one plate, put the silicon lid back on (and seal it tight) in the exact orientation.    
        + you don't want to introduce cross contamination.    
        + store the plate in -80ºC.   


## Part 2. DNA extraction    

### Materials    
1. Multi-channel pipettemen    
2. Autoclaved pipette tips and barrier pipette tips (various volumes)   
3. Disposable media trays    

### Procedures (mostly folow the kit protocol with some modifications)   
#### Experienced User Centrifugation Protocol    
##### NOTE:
+ Please wear gloves at all times     
+ BEFORE THE FIRST USE ONLY, Solution C5-D must be prepared. Add an equal amount of
100% Ethanol to Solution C5-D (for the 4 prep kit = 120 ml, or for the 12 prep kit = 360 ml).
Mix well. Put a check mark in the “ethanol added” box on the bottle cap label.

1. Add 750 µl of PowerSoil
-htp Bead Solution to the wells of the PowerSoil
-htp Bead Plate.    
    + use E1 Clip-tip 1250µl, program `Htp-750`    
2. Check Solution C1. If Solution C1 has precipitated, heat solution at 60ºC until the precipitate has
dissolved.
    + solution C1 contains SDS. If it gets cold, it will precipitate. Heating at 60C will dissolve the
SDS. Solution C1 can be used while it is still warm.     
3. Add 60 µl of Solution C1. Secure the Square Well Mat (from step 2) tightly to the plate.     
    + 
6. Place Bead Plate with mat securely fastened between 2 adapter plates (MO BIO Catalog# 11990)
and place on the 96 Well Plate Shaker (MO BIO Catalog# 11996). Reference the protocol provided
with the adapter plates for proper placement.
7. Shake at speed 20 for 10 minutes, remove plates and re-orient them so that the side closest to the
machine body is now furthest from the machine body and shake again at speed 20 for 10 minutes.
8. Centrifuge at room temperature for 6 minutes at 4500 x g.
9. Remove and discard the Square Well Mat. Transfer the supernatant to a clean 1 ml Collection Plate.
Note: The supernatant may still contain some soil particles.
10. Add 250 µl of Solution C2 and apply Sealing Tape to plate. Vortex for 5 seconds and incubate at 4C
for 10 minutes. Centrifuge the plate at room temperature for 6 minutes at 4500 x g. Remove and
discard Sealing Tape.
11. Avoiding the pellet, transfer entire volume of supernatant to a new 1 ml Collection Plate.
12. Apply Sealing Tape to plate. Centrifuge the plate again at room temperature for 6 minutes at
4500 x g. Transfer entire volume of supernatant to another new 1 ml Collection Plate.
13. Add 200 µl of Solution C3 and apply Sealing Tape to plate. Vortex for 5 seconds and incubate at 4C
for 10 minutes. Centrifuge at room temperature for 6 minutes at 4500 x g. Remove and discard
Sealing Tape.
14. Avoiding the pellet, transfer entire volume of supernatant to a new 1 ml Collection Plate.
15. Apply Sealing Tape to plate. Centrifuge the plate again at room temperature for 6 minutes at
4500 x g.
16. Transfer no more than 650 µl of supernatant to a 2 ml Collection Plate avoiding any residual pellet.
17. Add 650 µl of Solution C4 to each well of the plate.
18. Add a second 650 µl of Solution C4 to each well of the plate.
Note: It is safe to stop the protocol at this step and store the samples covered with
Sealing Tape (user supplied) at 4°C.
19. Pipet samples “up and down” to mix.
20. Place Spin Plate onto a new 0.5 ml Collection Plate.
21. Load approximately 650 µl into each well of the Spin Plate and apply Centrifuge Tape.
22. Centrifuge at room temperature for 3 minutes at 4500 x g. Discard the flow through and place the
Spin Plate back on the same 0.5 ml Collection Plate. Discard the Centrifuge Tape.
23. Repeat steps 20-21 until all the supernatant has been processed. Discard the final flow through.
24. Place the Spin Plate back on the same 0.5 ml Collection Plate.
25. Confirm that ethanol has been added to Solution C5-D (see step 1). Add 500 µl of Solution C5-D
to each well of the Spin Plate. Apply Centrifuge Tape to the Spin Plate.
26. Centrifuge at room temperature for 3 minutes at 4500 x g. Discard the flow through and place the
same 0.5 ml Collection Plate beneath the Spin Plate.
27. Centrifuge again at room temperature for 5 minutes at 4500 x g. Discard the flow through.
28. Carefully place the Spin Plate onto a Microplate. Remove Centrifuge Tape and discard.
29. Allow to air dry for 10 minutes at room temperature.
30. Add 100 µl of Solution C6 to the center of each well of the Spin Plate. Apply Centrifuge Tape.
31. Centrifuge at room temperature for 3 minutes at 4500 x g. Remove Centrifuge Tape and discard.
32. Cover wells of Microplate with the Elution Sealing Mat provided. DNA is now ready for any
downstream application. No further steps are required.
Prolonged storage at 4°C will result in the evaporation of eluted DNA. We recommend storing DNA
frozen (-20C or -80°C). Solution C6 does not contain EDTA. To concentrate the DNA see the Hints
and Troubleshooting Guide. 
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