Evan’s Capstone – eNotebook 1

Thursday – 1/21/2016

First day of Lab work on samples! Excited to get started on the study sites for the capstone project.

Samples from Swamp Creek (11 and 13) and May Creek (3 and 5) were pulled at 12:50 to be taken through the process of RNA Isolation as outlined in the Common Lab Protocols.

Some minor deviations due to conducting two different “sets” of two samples at a time and some overlaps when using the centrifuge with another lab member. Maybe try to figure out a better method of conducting multiple samples, maybe sets of 4 and then during the first incubation/centrifuging step set up another set of 4 samples?

During the first incubation of 15 minutes the second set of samples (5 and 13) incubated for 19 minutes while the first set of samples were in the centrifuge. After the 750 ul supernatant was pulled off and placed in a new snap-cap tube these two samples also rested for 5 minutes while waiting on the final ethanol washing step for the first set of samples (3 and 11). The first set of samples also had to be set to rest for 12 minutes before the final ethanol centrifuge, the ethanol supernatant was pulled off and the sample was set capped to ensure the ethanol did not evaporate off completely before the second 400 ul of ethanol could be added and the final washing step could be conducted.

The first set of samples were capped and held in the normal refrigerator until the second set was finished so we could bring them to the Nanodrop machine.

We set up the machine by first cleaning off the upper and lower lenses, placing 1 ul of clean water and initializing the nucleic acid option then cleaning the lenses and repeating the step again choosing the blank option. The lenses were cleaned in between each 1 ul sample and using the “measure” option.

Some errors occurred with new use of the machine (lower values or higher concentrations than expected, air bubbles in some situations, and some material not fully suspended on another sample), however the values from the successful runs of the machine were collected:

(ex. 3BMA is 3rd Sample, B for Cutthroat, MA for May Creek, SW for Swamp Creek)
3BMA – 260/280: 1.99; 260/230: 1.92; Concentration ng/ul: 1291.1
5BMA – 1.97, 1.80, 1111.3
11BSW – 1.96, 1.80, 745.7
13BSW – 1.98, 1.92, 1221.1

Completed samples placed back into the container and stored in -80 Celsius freezer at 5:15 PM and work concluded.

Good results and data collected so far and ready for work on Tuesday!

#rna, #salmon