Sample list is here (Google Sheet):
Samples were stored in Rack 1, Column 1, Row 1 in our -80oC.
Katie Lotterhos emailed the following note regarding some of the samples:
Hi all, As it shows in Alan’s spreadsheet, the sperm was diluted in a bit of seawater and flash frozen. We have had success centrifuging this (4000 rpm), removing the supernatant (seawater), and extracting the pellet of sperm.
We decided to send you the diluted sperm/seawater mixture because it is easier to transfer. Let us know how it works out with extraction. We have more that we can send you. Below I’ve added some notes from my tech last summer.
We also have data on the fertilization success of each male, and are still working on the larvae phenotyping for each male.>
Cheers, Katie
I spun the sample down using the large centrifuge in the core space (brought up to full speed at 4000 rpm). This concentrated the sperm nicely, which allowed me to draw off the extra sea water before adding the sperm sample. This caused the sperm to be very ‘snotty’ which means it wouldn’t suck up into the pipette tip when trying to add it to the 1.5mL centrifuge tube with lysis buffer. Blood can sometimes have a very similar ‘snotty’, viscous texture, so what I’ve done previously and did today was cut the tip of a 1000mL rip odd to make the tip opening larger. I then pipetted the entire concentrated sperm sample, at volumes, at increments of 500 mL, into the 1.5mL centrifuge tube. **NEXT TIME** Perhaps add the diluted sperm sample to the centrifuge tube, spin down, remove supernatant salt water, then add the lysis buffer. In order for this to work, the original sperm sample must be less than 1.5mL.