Objective: The objective of this experiment is to optimize an extraction protocol for marine seagrass associated sediment microbial communities. This is a 2 day (September 13th and 14th) process part of a larger multi-day experiment which will work to adjust this protocol to maximize Nucleic acid output. It is adapted from Lever et. al 2015.

Broader Context

This Protocol will simultaneously be compared to the zymobiomics DNA/RNA extraction kit. by using one of these protocols we will develop a reliable method for the extraction of seagrass associated microbes for Marine sediments, which we will be able to use in future experimentation.

Protocol

Using the below protocol we will compare 2 sediment samples taken from the same site but treated with diffeent preservation methods. 3 replicats per sample will be tested (n=3). The first treatment involved the direct and immediate freezing of sediment samples on dry ice these samples will be annotated as F1-F3. in the second method, zymoshield was added to the samples and allowed to sit for about an hour before freezing on Dry ice, annotated as Z1-Z3,

1. Sample preparation

a) Thaw samples over ice and keep on ice until flash freezing begins

b)add 0.2 g sample to a pre-filled bead beating tube (lysis matrix B, 0.1mm silicate bead)

c) Add 500μl of a 0.04M solution Sodium Hexametaphosphate over ice, pipette up and down to mix

2. Lysis

a) add 500 μL lysis Solution I, gently pipette up and down to mix

b) Flash Freeze sample in liquid nitrogen or Dry-ice ( if unavailable use a -80 15-20 min)

c) Thaw sample

d) Bead beat for 30s

e) Incubate at 50°C in for 1 hour, using the thermomixer at 500 rpm

f) Flash Freeze sample in liquid nitrogen or Dry-ice

g) Thaw sample

h) vortex for 10 s

i) Incubate at 50°C in for 1 hour, using the thermomixer at 500 rpm

j) Add Lysis II

k) Flash Freeze sample in liquid nitrogen or Dry-ice.

l)Thaw sample

m) vortex for 10 s

n) Incubate at 65°C in for 1 hour, using the thermomixer at 500 rpm

3. Purification

a) Centrifuge 15 minutes at 10,000xg and 4°C.

b) Transfer supernatant to a clean microcentrifuge tube.

c) Add 1 volume of CI vortex at max speed for 10 s

d) Centrifuge for 10 minutes at 10,000xg and 4°C

e) Transfer aqueous supernatant to clean microcentrifuge tube

f) Repeat c-e

4. Precipitation

a) Add LPA (20μL/ml final concentration) to sample and homogenize.

b) Homogenize sample with 0.1 volumes of 5 M NaCl solution followed by 1.5 volumes of isopropanol using a vortex, ensure samples look well combined

c) Incubate in the dark at -20°C for minimum 2h or overnight.

d) Centrifuge at 1400xg for 30 minutes at 4°C then pipette off the supernatant to leave the pellet, supernatant can be discarded

e) To remove isopropanol, pellets precipitated by isopropanol-containing solution are washed once with 100 microlitres 70% ethanol,centrifuged for 10 min at 14,000×g,

f) following centrifugation dry sample using the speed vac. turn off any temperature settings. us D-AQ mode, it should take 30 - 40 minutes

g) Dissolve pellet in 200μL of nucleotide free water. Vortex to dissolve.

5. Clean-up: Fraction sample for DNA and RNA (100μl/ tube) follow the below protocol for DNA and RNA

DNA	RNA
1.add 500μl of Binding Buffer H to each sample.	1. prepare the appropriate volume of buffer H by adding 10μl of betamercaptoethanol to each 1 ml added. ex. for 6 samples add 15μl to 1.5ml; addd 250 microlitres to each sample.
2.Vortex the sample to mix well	2. vortex the sample
3.assemble a column and collection tube for each sample	3. add 200μl of 96-100% EtOH
4.apply the 600μl of sample to the column	4. vortex for 10 seconds
5.centrifuge for 1 minute	5. apply the full volume of the sample to the spin column
6.Discard Flow Through	6. Centrifuge for 1 minute,
7.Place spin column back into the collection tube and add 500μl of Wash Solution K	7. Discard Flow through
8.centrifuge for 2 minutes at 14,000xg	8. place spin column back into the collection tube and add 500μl of Wash solution K
9.Discard flowthrough and centrifuge for an additional minute, ensure the column is completely dry	9. centrifuge for 1 minute ensuring the entire volume has passed through the tube (spin an additional minute if needed
10.discard flow-through and collection tube	10. discard flow through
11.Insert column into the elution tube	11. place spin column back into the collection tube and add 500μl of Wash solution K
12.Add 50μl of elution Buffer L	12. centrifuge for 2 minute ensuring the entire volume has passed through the tube (spin an additional minute if needed
13.centrifuge for 2 minutes at 200xg followed by 1 minute at 14,000xg	13. discard collection tube with flow through
14.Add an additional 50μl of Buffer L	14. place column into an elution tube and add 25μl of elution buffer L
15.Centrifuge for 2 minutes at 200xg followed by 1 minute at 14,000xg	15. centrifuge for 2 minutes at 200xg followed by 1 minute at 14,000xg
—	16.add an additional 25μl of elution buffer L
—	17. centrifuge for 2 minutes at 200xg followed by 1 minute at 14,000xg

Raw Data

Nano Drop Concentrations:

Sample	DNA conc.	DNA 260/280	RNA conc.	RNA 260/280
F1	12.1	1.78	29.8/27.9	1.59/1.56
F2	6.9	1.66	9.9/9.4	1.6/1.42
F3	8.1	1.95	10.0	1.66
Z1	3.4	1.59	3.9	1.32
Z2	3.7	1.41	5.6	1.69
Z3	2.4	1.65	3.0	1.61

Bioanalyzer readings:

Sample| Conc. ——-|—- F1 F2 F3 Z1 Z2 Z3

Data Outputs

If relevant, larger products from data sets may be shared here.

Concluding Thoughts

Protocol modifications.

• Zymo containing sediment sample is far more dificult to move into the bead beating tubes. in future sample prep I’d reccomend a lower volume addition if that ends up being the more impactful preservation method.

• Step 1C. the addition of 500 microlitres is to large a volume. I would reccomend the addition of about 200 for the next round.