Sperm DNA Extractions Part 2
Isolating and quantifying C. virginica sperm DNA
Yesterday I prepared two C. virginica sperm samples for overnight lysis. Today I’ll continue with the E.Z.N.A. Mollusc Kit to isolate DNA, then use the Qubit to quantify my yield. Hana, an undergraduate biology major, shadowed me while I did labwork! I’m hoping I can get her involved in helping with my labwork in the next few weeks.
Step 7. Remove samples from the heat block and add 350 µL of choroform:isoamyl acholol (24:1) to each sample. Vortext the samples to mix. Set the heat block to 70ºC.
Step 8. Centrifuge 10,000 x g for 2 minutes at room temperature.
- While centrifuging, I labelled two additional tubes for Step 9 with the sample name and “Aq” to designate that this was the aqueous phase (ex. 12s Aq)
Step 9. Transfer the upper aqueous phase to a clean 1.5 mL microcentrifuge tube. Take note of the quantity transferred. Avoid the milky interface containing contaminants and inhibitors.
- The milky interface was really easy to spot! I forgot I had my phone with me so I didn’t take any pictures, but I will next time.
- Both samples had 350 µL of the upper aqueous phase
Step 10. Add MBL Buffer in the same amount as the volume of the aqueous phase transferred in Step 8. Add 10 µL of RNase A to each sample, then vortex at maximum speed for 15 seconds.
- I added 350 µL MBL Buffer to each sample
Step 11: Incubate the samples at 70ºC for 10 minutes.
- The original protocol specifies this should be done in a water bath, but Sam said it was fine if I just used a heat block.
- The samples were left at room temperature for 13 minutes while I waited for the heat block to reach 70ºC
Step 12. Cool the sample to room temperature.
- I let the samples sit in the tube rack for 10 minutes to reach room temperature.
- While cooling, I pipetted 350 µL of the Elution Buffer into a 1.5 mL centrifuge tube, then placed it on the 70ºC heat block. I need the buffer at 70ºC for the final elution.
- I also labelled HiBind DNA Mini Columns with sample names while waiting for samples to cool.
Step 13. Add one volume 100% ethanol. Vortex at maximum speed for 15 seconds.
- I added 350 µL 100% ethanol to each sample.
Step 14. Insert a labelled HiBind DNA Mini column into a 2 mL Collection Tube. Transfer 750 µL of sample from Step 12 (including any precipitate) into the column.
- I did not have any precipitate.
Step 15. Centrifuge at 10,000 x g for 1 minute. Discard the filtrate and place the column back in the collection tube. Repeat Step 14 and 15 with until al of the sample has been applied to the spin column.
- I only needed to repeat these steps once more.
Step 16. Discard the collection tube. Place the spin column into a new collection tube and add 500 µL HBC Buffer to the column. Centrifuge at 10,000 x g for 30 seconds.
Step 17. Discard the filtrate and reuse the collection tube. Add 700 µL DNA Wash Buffer and centrifuge the samples at 10,000 x g for 1 minute. Repeat this step once more for a second DNA Wash Buffer wash step.
Step 18. Centrifuge the empty column at maximum speed for 2 minutes to dry the membrane.
Step 19. Place the spin column in a clean 1.5 mL microcentrifuge tube. Add 50-100 µL of the pre-heated Elution Buffer to the membrane and let it sit at room temperature for 5 minutes.
- I added 50 µL of buffer to each sample to get a more concentrated sample.
Step 20. Centrifuge at 10,000 x g for 1 minute. Repeat Step 19 and 20 once more for a second elution step.
- For the second elution, I used the eluate from the first elution instead of adding new elution buffer. I hoped this would increase my yield and concentration without changing my elution volume.
Step 21. Obtain dsDNA BR standards from the fridge.
Step 22. Prepare the master solution, using a 1:200 ratio of dye to dsDNA BR buffer. Each standard and sample needs 200 µL of solution.
- I had two standards and two samples, so I needed 800 µL of solution.
- I prepared 880 µL of solution, using 875.6 µL buffer and 4.4 µL dye (880 µL solution * 0.5 / 100 = 4.4 µL dye; 880 µL solution - 4.4 µL dye = 875.6 µL buffer).
Step 23. Pipet 200 µL master solution into each Qubit assay tube.
Step 24. Add 10 µL of the correct standard to the standard assay tube. Add 5 µL of sample to the sample tube. Vortex the tubes for 2-3 seconds, then incubate at room temperature for 2 minutes.
Step 25. Use Qubit to quantify yield
Table 1. Sample ID, concentration, and total DNA yield.
|Sample||Concentration (ng/µL)||Total DNA Yield (ng in 45 µL total)|
My two samples had very different concentrations! I think I was able to get enough DNA for whole genome bisulfite sequencing out of these samples. Since Sam isn’t in today for me to confirm, I’ll comment in this issue and continue with labwork tomorrow.
- Thursday: Pulverize remaining samples and start overnight incubation
- Friday: Finish isolating DNA from all samples and quantify with the Qubit