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|
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.