Sperm DNA MBD Enrichment Day 7

MBD-Biotin wash and binding

Before I started labwork in earnest, I prepared 10 mL of 1X Bind/Wash Buffer with 2 mL of 5X Bind/Wash Buffer and 8 mL of DNAse-free water. I then found the rotating mixer (FTR 213 fridge) and magnetic rack (FTR 209 drawer 43). I also updated this spreadsheet with my final DNA volumes and concentrations after shearing and quality checks. I have ten viable samples (not processing 31 due to low yield), six of which I’m able to use 4 µg of DNA from. I decided to process five samples today that I can use 4 µg of DNA from: 6, 7, 12, 13, and 23. The other five samples all require variable input volumes, so I can process them in a second batch. Working with five samples at a time also reduces the amount of tubes I’ll juggle when eluting DNA fragments.

I started the MethylMiner protocol stopped when incubating the DNA, dynabeads, and MBD-Biotin protein together. The reagent volumes I used in several steps, if not specified below, can be found here.

Step 1: Prepare beads

  • Labelled ten 1.7 mL centrifuge tubes with sample number and “B” (ex. 6 B)
  • Obtained Dynabead stock from 4ºC fridge
  • Resuspended beads by gently pipetting up and down (slow and only to the first stop)
  • Added 10 µL of beads for each µg of DNA to each centrifuge tube
  • Brought volume in tube up to 100 µL with 1x Bind/Wash Buffer, and gently mixed tube contents by pipetting
  • For each tube (one tube at a time):
    • Placed tubes on a magnetic rack for 1 minute to concentrate beads
    • Kept tube in rack and removed supernatant. It’s important to not touch the beads in this step!
    • Immediately removed tube in the rack and added 100 µL of 1X Bind/Wash Buffer to resuspend beads. The beads cannot be left dry for more than a minute.
    • Repeated this procedure once more for a total of two times.

Step 2: Bind MBD-Biotin protein

  • Labelled ten 1.7 mL centrifuge tubes with sample number and “P” (ex. 6 P)
  • Obtained MBD-Biotin Protein from -80ºC freezer (rack 12, column 3, row 2)
  • For each µg of input DNA, I added 7 µL of MBD-Biotin Protein
  • Topped off volume to 200 µL with 1X Bind/Wash Buffer, gently pipetting to mix
  • Transfered MBD-Biotin protein tube contents to corresponding “B” tube
  • Placed all “B” tubes on a rotating mixer for one hour at room temperature
    • Start: 10:47 a.m., end: 11:47 a.m.

Step 3: Wash MBD and beads

  • For each tube:
    • Placed tubes on a magnetic rack for 1 minute to concentrate beads
    • Kept tube in rack and removed supernatant. It’s important to not touch the beads in this step!
    • Immediately removed tube in the rack and added 100 µL of 1X Bind/Wash Buffer to resuspend beads. The beads cannot be left dry for more than a minute.
    • Placed tubes back in rotating mixer for 5 minutes at room temperature
    • Repeated this procedure twice more for a total of three times.
  • For each tube:
    • Placed tubes on a magnetic rack for 1 minute to concentrate beads
    • Kept tube in rack and removed supernatant. It’s important to not touch the beads in this step!
    • Immediately removed tube in the rack and added 100 µL of 1X Bind/Wash Buffer to resuspend beads. The beads cannot be left dry for more than a minute.

Step 4: Add DNA

  • Set up rotating mixer in the 4ºC fridge in FTR 213
  • Diluted samples to 25 ng/µL with DNAse-free water
    • Sample 7 needed to be diluted with ~800 µL of water so I needed to transfer the sample from the small shearing tube to a 1.7 mL centrifuge tube
  • Labelled ten 1.7 mL centrifuge tubes with sample number and “DNA”) (ex. 6 DNA)
  • Added 100 µL of 5X Bind/Wash Buffer to each tube
  • Added designated sample DNA volume from calcuations to each “DNA” tube
    • Used all of sample 6 and 12
  • Topped “DNA” tube volumes to 500 µL with DNAse-free water
    • And…I messed this up a little bit. Before I topped off the volumes, I realized I added 100 µL of 1X Bind/Wash Buffer and not 5X Bind/Wash Buffer! Adding 100 µL of 1X Bind/Wash Buffer means that I added 20 µL of 1X Bind/Wash Buffer and 80 µL of DNAse-free water. I added 80 µL of 5X Bind/Wash Buffer to each “DNA” tube. I subtracted 80 µL from the amount of DNAse-free water needed to top off the volumes to 500 µL, then added that! Another instance of a potentially-disasterous-but-luckily-not lab struggle.
  • Transfered “DNA” tube contents to corresponding “B” tubes
    • When I transfered the “DNA” tube contents to the “B” tube contents, the measured volume of all the tubes was 500 µL. My calculations in the previous step worked out.
    • I spilled a little bit of sample 13 when transferring the contents. Only a little bit leaked out of the pipet and when I looked at the volume of liquid in 13 B vs. 23 B, they looked very similar
  • Mixed tubes on a rotating mixer overnight at 4ºC
    • Placed the tubes in the mixer at 1 p.m.

Going forward

  1. Label six sets of 1.7 mL centrifuge tubes for non-captured DNA, washes, and elutions
  2. Make 10 mL more of the 1X Bind/Wash Buffer
  3. Complete elutions and ethanol precipitations
  4. Process second batch of C. virginica sperm samples
Written on August 12, 2020