Virginica Gonad DNA Extractions Part 10
Testing protocol for sperm samples
Now that I’m done with the female gonad samples, I need to extract DNA and RNA from sperm samples. According to this spreadsheet, the samples are in 100 µL of a seawater solution. I wasn’t sure if I should attempt to pellet the sample and remove the seawater. Based on Sam’s feedback this issue, I should proceed with the protocol for cells in suspension or other liquids. So that’s what I did!
Methods: Sample Preparation
Step 1: Prepare for extractions.
- Label 3 sets of tubes RNase-free centrifuge tubes per sample: one for the sperm sample, one for final RNA storage, and one for final DNA storage.
- Label spin columns
- Obtain samples from -80ºC freezer
Step 2: Obtain 50 µL of sperm sample in a seawater solution and pipet into a labelled centrifuge tube.
- I chose 50 µL of sample since I thought it would allow me to test the protocol without using all of the sperm sample sent
- After bringing the sample to room temperature, I pipetted up and down to mix and took out 50 µL. I noticed that there were small pieces of tissue in the solution, but it was mainly cloudy liquid. I assume this means that there may be some connective tissue in the samples.
Step 3: Add 4 volumes of DNA/RNA Lysis Buffer and mix
- I added 200 µL of Lysis Buffer to each sample for a total volume of 250 µL
- For sample 12, the pipet tip got stuck and the liquid wouldn’t come out! I ended up using ethanol to clean a pair of scissors, then cutting off the pipet tip above where the liquid was to break the vacuum seal. I lost some liquid, but I had more than 200 µL of liquid in the tube. Since I mixed the buffer while adding it to my sample, I don’t think I only lost sample or buffer when I lost liquid.
- Vortexed for 10 pulses
Methods: Sample Purification
I also grabbed DNAse from the -20ºC freezer and put the nuclease free water on a heat block at 95ºC.
Step 4: Transfer the sample into a IC-MX spin column in a collection tube and centrifuge.
- Kept the labelled spin columns for DNA extractions
- Saved the flow-through for RNA extractions
Step 5: For RNA only, add an equal volume of 95-100% ethanol to the flow-through and mix by pipetting. Transfer the flow-through into a new IC spin column in a clean collection tube. Centrifuge and discard the flow-through.
- Grace took on the RNA tubes!
- Added 250 µL of EtOH and mixed
Step 6: For the RNA IC spin columns, perform a DNAse I treatment
- Grace did this!
- Added 400 µL of the DNA/RNA Wash Buffer to each column and centrifuged, then discarded the flow-through.
- Made a DNase REaction Mix with 5 µL of DNase I and 35 µL DNA Digestion Buffer for each sample.
- Added the mix to the column matrix and incubated it at room temperature for 15 minutes before proceeding.
Step 7: Add 400 µL DNA/RNA Prep Buffer to the column. Centrifuge and discard the flow-through.
Step 8: Add 700 µL DNA/RNA Wash Buffer to the column. Centrifuge and discard the flow-through.
Step 9: Add 400 µL DNA/RNA Wash Buffer to the column. Centrifuge at 16,000 rcf for 2 minutes. Transfer the column to a new microcentrifuge tube.
Step 10: Add 15 µL DNase/RNase-Free Water to the column. Incubate at room temperature for 5 minutes. Centrifuge to elute the DNA and RNA.
- Added 15 µL of 95ºC nuclease-free water to the column
- Samples were centrifuged for 1 minute
Methods: Sample Quantification
Step 11: Prepare the master solution using a 1:200 ratio of buffer to dye. For each sample and both standards, 200 µL of master solution is needed.
- RNA: Grace did this!
- DNA: Pre-mixed master solution for the Qubit dsDNA HS kit (aka my favorite thing)
Step 12: For each standard’s designated Qubit assay tube, add 10 µL of the correct standard and 190 µL of the master solution.
Step 13: For each sample’s designated Qubit assay tube, add 1 µL of the sample and 199 µL of the master solution.
Step 14: Vortex all Qubit assay tube for 2-3 seconds at maximum speed. Incubate tubes at room temperature for two minutes.
Step 15: Use Qubit to quantify RNA and DNA yield in each sample tube.
- DNA: S1 = 80.03, S2 = 39903.70; RNA: S1 = 83.23, S2 = 1864.03
- Updated yields can be found here
- I’m worried that I won’t have enough RNA for RNA-Seq libraries with 50 µL of starting volume. It’s time to get some quotes and figure out how much RNA is needed at different facilities. I could also use the remaining 50µL of each sample for RNA exractions, but we’ll see.
- Extract male samples and check yields
- Send DNA and RNA for library preparation and sequencing