Objective:

This is the first use of the ZymoBiomics DNA/RNA Prep minikit. We are hoping this kit will prove to be a more efficient alternative for Sediment extractions that we can use for metagenomic analyses and metabolomics. the purpose of this experiment was to test the protocol per the manufacturers instructions on our marine sediment samples collected in Bodega Bay

Broader Context

Both this extraction and the previous entry were done on sediments collected from Bodega in July of 2021. We hope to be able to optimize one or both of these protocols in order to use on these samples containing high volumes of humic substances.

Protocol

In the future Wash Buffer will require 104ml Ethanol added to the concentrate. For the purposes of the sample kit, the Buffer comes ready to Use. ### 1. Sample Preparation a) add approximately 0.2 g of sediment if stored frozen, then add 750μL of Zymoshield.

b) For this Experiment Samples were already frozen in Zymo shield, so the sample Was vortexed, and then pipetted up and down to disturb fallen sediment. 750 μL total volume was added to the provided tube with lysis matrix.

c) Lysis tubes were bead beat in the Fast-Prep 24-5G at 6.5m/s for 1 minute with a 5 minute rest for 5 cycles.

d) Centrifuge at 14,000xg for 30 seconds and transfer 400 μL to a clean microcentrifuge tube.

e) add 2 volumes of Lysis buffer (800 μL) to your sample and vortex to mix

f) Transfer 700 μL your sample to the Spin column with the yellow ring and spin at 14,000xg for 30 seconds. you’ll need to repeat this a second time to capture the last 100 μL of your sample Save Your Flow through!!!!! your RNA is in the flow through and your DNA is bound to the column.

g) place your column in a new collection tube and set to the side

2. RNA digestion.

a) take your flow though from Step 1f) and transfer it to a clean microcentrifuge tube.

b)add an equal volume (in this case about 800 μL of Ethanol). Vortex the solution

c) transfer the sample into the green spin column and centrifuge at 14,000xg for 30 seconds( you’ll need to do three rounds of this 2x 700 μL plus one for the remaining volume.)

d)Discard the flow through

e) prepare the you DNaseI solution. add 5 μL of reconstituted DNaseI to 75 μL of DNase digestion buffer per sample. the DNaseI should be stored in the -20 °C when not in use.

f) add 80 μL of you DnaseI buffer mixture directly onto the spin column and let incubate for 15 minutes at room temperature.

### 3. Washing and Elution a) get the DNA samples columns which you’ve put to the side, along with you RNA Samples. Proceed with all further steps for both DNA and RNA columns simultaneously

b) add 400 μL of DNA/ RNA Prep Buffer to the spin column.

c) centrifuge for 30 seconds at 14,000xg and discard flow through

d) add 700 μL of DNA/RNA Wash buffer, centrifuge for 1 minute and discard the flow through.

e) add an additional 400 μL of wash buffer to the column and centrifuge at 14,000xg

A Note on step 3e): According to the manufacturers protocol the centrifugation time for this step should be 2 minutes. However, Upon moving to the next step even though it appeared that all liquid had migrated through the column, eluate volumes appeared uneven. my suspicion is that while the liquid had begun to pass through the column, some of it remained trapped within the column. Therefore I would reccomend discarding the flowthrough anf centrifuging for an additional minute before moving on.

f) add 100 μL of Nuclease free water to the column and let incubate at room temperature for 5 minutes

g) While your columns are incubating, add 600 μL of HRC prep solution to the III-HRC column and centrifuge at 8,00x6 for 3 minutes.

h) Place the spin column in a clean microcentrifuge tube and centrifuge you sample columns at 14,000xg for 30 second

i) transfer you 100 μL of eluate to the III-HRC column and place the column in a clean microcentrifuge tube.

j) Centrifuge the Sample-containing III-HRC column at 16,000xg for 3 minutes

k) Store your samples at -80 °C until they are ready for downstream processing. A clean kit should not be needed.

Raw Data

Nanodrop Concentrations

Sample	DNA conc.	DNA 260/280	RNA conc.	RNA 260/280
K1	8.4/8.7	1.92/2.16	7.3/6.6	2.18/2.14
K2	7.1/8.9	2.13/1.98	8.9/7.6	2.33/2.27

Concluding Thoughts

All modifications to the manufacturers protocol have been noted above. Samples are currently being stored at -80 °C. I will update this entry with Nano-drop concentrations. Due to the uneven eluate volumes I am concerned about buffer contamination, particularly in the RNA samples. Based on the results of the Nanodrop, I may decide to run the Norgen Clean Kit on the samples if the ratios seem off.

Overall I found this protocol to be quite efficient, and furthermore, I think its far easier to add the zymo storedd sediment as a liquid volume rather than trying to measure .2g of sediment.