Yaamini’s Notebook: Sperm DNA Extractions Part 5

Requantifying C. virginica sperm DNA extractions

I thought I was done with my sperm DNA extractions, but turns out I did my Qubit quantifications incorrectly (thanks for reading my notebook and pointing that out Sam). I was pipetting 200 µL of the master solution into each Qubit assay tube, when I should have had 200 µL total (master solution AND sample) in the assay tube. I popped into the lab to quickly finish this off!

Quantificiation

Step 23. Obtain dsDNA BR standards from the fridge. Label assay tubes for each sample and two standards.

  • There were some off-brand assay tubes next to the Qubit. I figured they were for use with the Qubit, so I used them for some of my samples once I was out of Qubit brand tubes.

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 eleven samples, so I needed 2600 µL of solution.
  • I used 2786 µL buffer and 14 µL dye (2600 µL solution * 0.5 / 100 = 14 µL dye; 2800 µL solution – 14 µL dye = 2786 µL buffer).

Step 25. For each standard, pipet 190 µL master solution and 10 µL of the standard into the assay tube.

Step 26. For each sample, pipet 199 µL master solution and 1 µL of the sample into the assay tube.

Step 27. Use Qubit to quantify yield

Results

Table 1. Sample ID, concentration, and total DNA yield. Standard 1 read at 257.82, and Standard 2 at 26678.08. Asterisks indicate the sample tube used was not a Qubit brand tube.

Sample ID DNA Concentration (ng/µL) Final Volume (µL) Total DNA Yield (ng)
L18A0006s 96 44 4224
L18A0007s 432 43 18576
L18A0009s 46.4 44 2041.6
L18A0012s* 73.6 44 3228.4
L18A0013s 186 44 8184
L18A0023s 344 43 14792
L18A0031s 9.24 44 406.56
L18A0048s* 25.8 43 1109.4
L18A0057s* 163 44 7172
L18A0059s* 55.6 44 2446.4
L18A0063s* 99.4 44 4373.6

Going forward

  1. Prepare these samples for whole genome bisulfite sequencing!

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