Objective: This is the third round of experimentation on this project with the goal of further improving the method of extraction on marine sediments with high humic contents. first round data can be found in the post from Sept. 14th

Broader Context

The samples used here were part of the initial sample collection from the scouting trip to Bodega bay in late july. successful optimization of this protocol will allow us to simultaneously collect DNA and RNA from a single sample to be used in multiple downstream analyses such as simultaneous metagenomics and metabolomics

Protocol

The protocol used followed the same protocol as that from the experiment on Sept. 14th with the following modifications.

• In step 1C liquid weight was used as an approximation for gram weight. 200μL of sediment regardless of wether it was stored in Zymo research’s DNA/RNA Shield or frozen directly.
• In step 3 the number of CI washes was increased from 2 to 3 washes.
• In step 4e was repeated for a total of 2 EtOH washes. EtOH was pipetted off each time before drying down the sample in the speed vac for 10 minutes.
• the DNA aliquout from S13 was not run through the clean kit, due to a lack of columns.

  Raw Data

Concluding Thoughts

• increasing number of washes does seem to slightly improve quality however samples arestill highly contaminated
• will run samples from frozen through zymo kit to check success after dilution -in future rounds, a wash with PCI may be tried as the third wash.