Evan’s Capstone – eNotebook 3

Thursday – 1/28/2016

Plan for today changed slightly, just continuing more RNA isolations in the hopes of getting them all done by next week and continuing on to the next part of the project. Operation running smoothly now, essentially had no downtime during sample work with the centrifuge running with either set of samples at a time. Also started work on two new creeks now that the first 6 samples of Swamp Creek and May Creek are finished!

First set of samples consisted of 11 and 13 of Issaquah (#BIS) in addition to 13 and 14 of Harris (#BHA) and were pulled at 11:50 AM. The second set was 12 and 15 of Harris with 3 and 17 of Issaquah which were pulled at 12:10 PM.

A few more deviations this time due to balancing centrifuge time with my supervisor; Set 1 rested for 4 additional minutes after Step 18 and then after Step 21 rested for longer than anticipated (8 minutes) and was re-centrifuged for 3 minutes to ensure integrity of pellet. Set 2 rested after Step 13 for 4 additional minutes and also rested after Step 18 for 2 additional minutes. Sample 14BHA ended up being re-suspended in 25 ul of DEPC-treated water instead of the usual 50 because of a small liver sample and subsequently a small pellet size.

Nanodrop results for the day:

12BHA – 260/280: 1.97, 260/230: 2.01, Concentration 720.6
13BHA – 1.98, 2.26, 1574.7
14BHA – 1.74, 2.08, 391.0 (~)
15BHA – 1.94, 2.09, 774.5

3BIS – 1.95, 2.09, 844.6
11BIS – 1.96, 1.90, 644.8
13BIS – 1.93, 2.27, 977.6
17BIS – 1.82, 1.57, 241.1 (~)

The tilde is to represent the two samples I am questioning on using given the less than optimal numbers (both have numbers lower than 1.8 which is what I’ve been looking for). In hindsight, 17BIS had a small liver sample size similar to 14BHA and given how low the concentration is, it probably would have been smart to limit the re-suspension to 25 ul for it also. I’m not sure if the small liver sample size is to blame for the low Nanodrop numbers? I will need to consult with someone about what to do with them.

All samples were placed in the -80 freezer at 4:40 PM following the Nanodrop results. My goal for next week will be to get at least 6 samples from each creek finished so that they can be brought through the next steps and start to see some results. May or may not have to pull an extra sample for Harris and Issaquah given the two “maybes” from today; We’ll see!

#rna, #salmon