Laura’s Notebook: Oly DNA Isolation, take 3

Soaked mortar, pestle, metal spatulas in 10% bleach/DI water (100mL Clorox bleach, 900mL DI water) for ~15-20 minutes. Rinsed with DI water, let dry then covered individually in foil and autoclaved at 121C for 20 minutes.

Next day, put mortar & pestles in -20 for a copule hours. Picked up 6L of liquid nitrogen – note this is availabe in the Bagley, the Chem supply store, not in the J-wing of the microbiology building where we get dry ice.

Got larvae and mantle tissue out of the -80 for my test run, on the following groups:

DNA VIAL # SAMPLE # Treatment TISSUE TYPE
test10 27-A HL-10-Low Frozen larvae
test11 60-A HL-10-Low Frozen larvae
test12 73-A HL-10-Low Frozen larvae
test13 59-A HL-6-Ambient Frozen larvae
test14 HL-10-12 HL-10-Low Frozen mantle
test15 HL-10-10 HL-10-Low Frozen mantle
test16 HL-6-18 HL-6-Ambient Frozen mantle
test17 HL-6-20 HL-6-Ambient Frozen mantle

Used E.Z.N.A. Mollusc DNA Kit NOTE: we have 8 mortar/pestle sets in our lab. Can’t do more than 8 samples / day.

Labeled and weight 1.5mL microcentrifuge tubes.

Held samples on wet ice until homogenized. For larvae, they were all at the bottom of the tube. I inverted the tube and tapped vigorously to get as much onto the mortar as possible; also used autoclaved toothpicks to try to get more out. Probably shouldn’t do this next time – find another small item to scrape tissue out of tube. Curious if I can add ethanol or something to get the larvae into solution, then pour into mortar? Will ask the lab.

Poured LN2 into mortar with pestle also in; grouned tissue down. Used metal spatula to scrape frozen tissue dust into pile, carefully transferred into new 1.5mL microcentrifuge tube. This was also very difficult, as the spatula just barely fit into the tube, and if the tissue started to melt at all it would stick to the spatula. Need a better tool for transferring tissue – smaller spatula with round tip? Also, sterilized the spatula by soaking in bleach, then 2 DI rinses, between every-other sample (used 2 spatulas). Adequate? Will ask lab.

Need no more than 30mg for each sample; weighed tubes+samples. For samples with >30mg, labeled and weighed new tubes and either transferred ~30mg into newly labeled tube or removed a bit of sample.

Added 350uL buffer, and 25uL proteinase K. Floated in 60C water bath. Kit says to incubate for 30 mins – 4 hours.

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Grace’s Notebook: Updates Podcast Dia

Podcast

Today Steven and I recorded a little bit of us talking about the spreadsheet and how I’ll be doing the RNA isolating. What I am going to work on is an episode, or several short episodes, about the background of the project: bitter crab disease; Tanner crabs; Juneau, etc. I also am going to make a quick little intro that we can put at the beginning of all the episodes. I want to make one weekly, or at least release one weekly. And I would like to have some way to make it more easily understandable to people like my mom, or my friends. To do that I think I just have to make sure I explain what I’m talking about more. Obvoiusly I shouldn’t be putting too much time into it because the main focus is on the research itself, but I’m excited and looking forward to learning more about this outreach tool and communication strategy.

DIA Analysis

I looked at the DIA analysis of the 2015 oysterseed. I have no idea what I’m looking at anymore and am getting confused. I read a Skyline tutorial that Emma recommended I look at in order to learn more about how to tell if Skyline is picking peaks well (tutorial) but I am not fully grasping it. So, Emma is going to stop by on Monday to show me what’s what!

RNA Isolation

I am going to pick samples such that I’m following individuals throughout the entire project and within treatment groups. I’ll begin next week and will keep track of what I’m doing.

Crab data sheet

I re-organized the data sheet such that it is machine-readabe. Per Steven’s suggestion, I made a unique ID for each sample following this pattern:

FRP#_Tube#_SampleDay#

Example: 6116_76_9

6116 is the unique crab ID number (there are three samples with 6116_ at the start, because this data includes only crabs that survived the project, and thus have three samples.

76 is the tube number that the hemolymph sample is in.

9 is the sample day number.

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