Yaamini’s Notebook: Sperm DNA Extractions Part 4

Finishing C. virginica sperm DNA extractions

Last day of sperm DNA extractions! Kaitlyn helped me isolate DNA from the samples I incubated overnight so we sped through the protocol.

DNA Isolation

Step 9. Remove samples from the heat block and add 350 µL of choroform:isoamyl acholol (24:1) to each sample. Vortex the samples to mix. Set the heat block to 70ºC.

  • I removed the samples at 8:50 a.m., so the incubation ran for about 22 hours.

Step 10. Centrifuge 10,000 x g for 2 minutes at room temperature.

  • While centrifuging, I labelled additional tubes for Step 11 with the sample name and “Aq” to designate that this was the aqueous phase (ex. 48s Aq)

Step 11. 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.

  • This was tricky for 57s and 63s. I sucked up some of the milky interface as I was pipetting and tried to not transfer it to the microcentrifuge tube, but some got in there.
  • Volume upper aqueous phase transferred:
    • 300 µL: 9s, 48s
    • 350 µL: 6s, 59s
    • 400 µL: 7s, 13s, 57s, 63s
    • 450 µL: 23s

Step 12. Add MBL Buffer in the same amount as the volume of the aqueous phase transferred in Step 11. Add 10 µL of RNase A to each sample, then vortex at maximum speed for 15 seconds.

  • Volume MBL Buffer added:
    • 300 µL: 9s, 48s
    • 350 µL: 6s, 59s
    • 400 µL: 7s, 13s, 57s, 63s
    • 450 µL: 23s
  • I FORGOT TO ADD RNASE TO MY SAMPLES. I didn’t realize this until it was too late, so I hoped it wouldn’t interfere with my DNA yields.

Step 13: Incubate the samples at 70ºC for 10 minutes on a heat block.

Step 14. Cool the sample to room temperature.

  • I let the samples sit in the tube rack for 10 minutes to reach room temperature.
  • I also labelled HiBind DNA Mini Columns with sample names while waiting for samples to cool.

Step 15. Add one volume 100% ethanol. Vortex at maximum speed for 15 seconds.

  • Volume MBL Buffer added:
    • 300 µL: 9s, 48s
    • 350 µL: 6s, 59s
    • 400 µL: 7s, 13s, 57s, 63s
    • 450 µL: 23s

Step 16. 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.

  • There was no precipitate in any of my samples.

Step 17. Centrifuge at 10,000 x g for 1 minute. Discard the filtrate and place the column back in the collection tube. Using the same column, repeat Step 16 with until all of the sample has been applied to the spin column.

  • I repeated Step 16 once more for all samples.

Step 18. 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 19. 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 20. Centrifuge the empty column at maximum speed for 2 minutes to dry the membrane.

Step 21. 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 22. 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.

Quantificiation

Step 23. Obtain dsDNA BR standards from the fridge.

Step 24. 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 nine samples, so I needed 2200 µL of solution.
  • Kaitlyn prepared 2220 µL of solution, using 2208.9 µL buffer and 11.1 µL dye (2220 µL solution * 0.5 / 100 = 11.1 µL dye; 2220 µL solution – 11.1 µL dye = 2208.9 µL buffer).

Step 25. Pipet 200 µL master solution into each Qubit assay tube.

Step 26. 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 27. Use Qubit to quantify yield

  • When I tried quantifying 7s and 23s, it said the yield was too high and I had to dilute my sample! Kaitlyn and I prepared two more assay tubes using only 1 µL of sample and got a viable Qubit reading. The final volumne of these samples is 44 µL instead of 45 µL.

Results

Table 1. Sample ID, concentration, and total DNA yield. Standard 1 read at 237.36, and Standard 2 at 22789.76.

Sample ID DNA Concentration (ng/µL) Final Volume (µL) Total DNA Yield (ng)
L18A0006s 113 45 5085
L18A0007s 576 44 25344
L18A0009s 552 45 24840
L18A0013s 208 45 9360
L18A0023s 416 44 18304
L18A0048s 35.6 45 1602
L18A0057s 312 45 14040
L18A0059s 95.2 45 4284
L18A0063s 173 45 7785

Going forward

  1. Prepare these samples for whole genome bisulfite sequencing!

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Yaamini’s Notebook: March 2019 Goals

fun

No no small child, don’t worry. There is a spring break. And I’m even going on vacation! However, this means that I only have 19 days to finish up all of my March goals :0 But first, let’s review last month’s progress.

February Goals Recap:

Bypass:

  • Submitted my bypass application…AND GOT MY BYPASS APPROVED! That’s right. I’m a Ph.D student now :0

Virginica:

Gigas Broodstock:

  • Didn’t send samples for sequencing or determine if I can isolate RNA, but I did make a nifty flowchart.

March Goals

Virginica:

  • Finish gene product descriptions
  • Compare hypermethylated and hypomethylated DMRs and DMLs
  • Draft discussion
  • Finish sperm DNA extractions and send samples for whole genome bisulfite sequencing

Gigas Broodstock:

  • Figure out what to do with the isolated DNA…and do it.

Other:

  • Add new members to my committee
  • Schedule a committee meeting
  • Figure out when I’m taking my written exam
  • Polish my Ph.D proposal

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Yaamini’s Notebook: DML Analysis Part 25

DML-mRNA gene product information

I repeated my DMR-mRNA overlap product description procedure with the DML-mRNA overlaps. I then pored over my product summary file. I slightly regret manually processing the file in Excel instead of finding a nifty way to do it in bash because it took me over an hour…HOWEVER I learned some interesting things.

There were some gene products, like multidrug resistance-associated protein and heat shock 75kDa, that were prominent in the DML file but not present in the DMR overlaps. I think this has to do with the fact that the same coding region can have loci that are both hyper- and hypomethylated. Since DMRs are looking at methylation differences across 100 bp, some of these opposite effects “cancel out” and are not significantly differentially methylated.

Some coding regions had mulitple DMLs that were all hyper- or hypomethylated, but they had different methylation differences. It’s possible for there to be several DMLs in a coding region, but spread out such that it does not constitute a DMR. Since the mRNA coding regions include exons and introns, it may be valuable to consider exons and introns separately in all downstream analyses.

There were several gene products related to human cancers, which didn’t make any sense. I did not annotate the genome myself, but it would be worth learning how genome annotation happens just so I can understand these annotations (or know to ignore them).

Going forward

  1. Repeat this process with exon, intron, transposable element overlaps
  2. Compare genes with hypermethylated vs. hypomethylated loci and regions

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