Virginica Gonad DNA Extractions Part 4
Subsampling remaining female tissue
Methods: Sample Preparation
Step 1: Prepare for extractions.
- Label 3 sets of tubes RNase-free centrifuge tubes per sample: one for frozen tissue, one for final RNA storage, and one for final DNA storage.
- Obtain samples from -80ºC freezer and place on wet ice
- Since I only had four spatulas available to me, I decided to process samples in batches of 3-4. I grabbed one batch from the -80ºC at a time and covered the samples in wet ice to prevent samples from thawing too much.
Step 2: Cut and weigh no more than 10-15 mg of frozen tissue. Record weight of tissue used in extractions and place tissue in a new, labelled test tube.
- I tared the scale with a piece of weigh paper. I used a clean spatula with a sharp edge to cut the tissue in the tube. Once I got the weight I wanted, I transferred the tissue to the labelled centrifuge tube. The spatulas were washed in 200 mL of a 10% bleach solution, then rinsed with DI water after both samples were prepared.
- Some samples were very gamete rich (“milky”) so they were tricker to weigh. After getting the sample mass, I put the sample back in the tube the samples were mailed in so I could digest the milky gametes that I couldn’t put on the scale. I’ll need to transfer these samples to 1.5 mL centrifuge tubes after overnight incubation.
Table 1. Mass of samples used for DNA/RNA extractions. Samples that were too “milky” to transfer to 1.5 mL centrifuge tubes were kept in the mailing tubes after subsampling, meaning that I used all the sample in that tube. There were also samples that do not have any more remaining tissue after obtaining 10-15 mg of tissue.
|Sample ID||Weight Sent (mg)||Subsample Mass (mg)||Notes|
|29||16||9.0||All sample used|
|41||18||11.5||All sample used|
|53||18||10.8||All sample used|
|76||22||~5-6||Very milky, couldn’t measure accurately|
I put the subsampled and weighed tissues back in the -80ºC. Tomorrow, I’ll add DNA/RNA shield and digestion buffers to each sample and start an overnight incubation.
- Perform DNA and RNA extractions with subsampled tissues.
- Check DNA and RNA yield and quality for female samples
- Test extraction protocol for male samples
- Send DNA and RNA for library preparation and sequencing