Yaamini’s Notebook: Virginica MBDSeq Day 4

Rinse and repeat

Today I got really good at pipetting supernatant from tubes as they were on the magnetic rack, then adding liquid to the beads and placing the tubes on the rotating mixer. Maybe it’s because that’s all I really did………..

Step 0: Prepare for the day

  • Made 10 mL of the 1X Bind/Wash buffer using the same ratio as yesterday.
    • I used the same bottle of buffer I used yesterday and mixed everything. Then I realized there probably wasn’t a full 2 mL in that bottle since the pipet tip had lots of bubbles. I discarded the buffer I made, and remade it using 5X Bind/Wash Buffer from a new bottle. I labelled this bottle with the date and the volume I took from it.
  • Labelled six sets of 10 1.7 mL centrifuge tubes!
    • Sample Number + Wash 0 (ex. 31 W0)
    • Sample Number + Wash A (ex. 31 WA)
    • Sample Number + Wash B (ex. 31 WB)
    • Sample Number + Elution 1 (ex. 31 E1)
    • Sample Number + Elution 2 (ex. 31 E2)
    • Sample Number + Elution 3 (ex. 31 E3)
  • Got ice
    • Sidenote: the new ice machine is so fancy

Step 1: Remove non-captured DNA

  • Removed tubes from the rotating mixer at 4ºC
  • For each tube:
    • Placed tube on magnetic rack for one minute
    • Pipetted supernatant from “B” tube into corresponding “W0” tube, and placed “W0” tube on ice
    • Added 200 µL 1X Bind/Wash buffer to “B” tube
  • For each tube:
    • Placed tube on rotating mixer for 3 minutes
    • Placed tube on magnetic rack for one minute
    • Pipetted supernatant from “B” tube into corresponding “WA” tube, and placed “WA” tube on ice
    • Added 200 µL 1X Bind/Wash buffer to “B” tube
    • Repeated entire process once more
  • For each tube:
    • Placed tube on rotating mixer for 3 minutes
    • Placed tube on magnetic rack for one minute
    • Pipetted supernatant from “B” tube into corresponding “WB” tube, and placed “WB” tube on ice
    • Added 200 µL 1X Bind/Wash buffer to “B” tube
    • Repeated entire process once more, EXCEPT I added 400 µL of High Salt Elution Buffer to the “B” tube after pipetting the supernatant from the “B” tube and into the “WB” tube on the second round. I got the High Salt Elution Buffer from the 4ºC fridge

Step 2: Elute captured DNA

  • For each tube:
    • Placed tube on rotating mixer for 3 minutes
    • Placed tube on magnetic rack for one minute
    • Pipetted supernatant from “B” tube into corresponding “E1” tube, and placed “E1” tube on ice
      • During this step, I spilled some of sample 106 E1 as I was pipetting it from the “B” tube to the “E1” tube
    • Added 400 µL High Salt Elution Buffer to “B” tube
  • For each tube:
    • Placed tube on rotating mixer for 3 minutes
    • Placed tube on magnetic rack for one minute
    • Pipetted supernatant from “B” tube into corresponding “E2” tube, and placed “E2” tube on ice
    • Added 400 µL High Salt Elution Buffer to “B” tube
  • For each tube:
    • Placed tube on rotating mixer for 3 minutes
    • Placed tube on magnetic rack for one minute
    • Pipetted supernatant from “B” tube into corresponding “E3” tube, and placed “E3” tube on ice
    • Discarded “B” tubes

I finished this stage at 11:05, and took a break until 12:05 to eat lunch. I covered the samples with extra ice to keep them cool.

Step 3: Ethanol precipitation

Kaitlyn helped me with this step, which was nice since I had 60 tubes!

  • Obtained glycogen from -20ºC freezer
  • Added 1 µL glycogen to each tube (all W0, WA, WB, E1, E2 and E3 tubes)
    • I had sets W0, WB, and E2. Kaitlyn had the others
  • Added 1/10th sample volume of pH 5.2 3 M sodium acetate (made by Sam in March 2007) to each sample
    • I measured the contents of the W0 tubes with a pipet, since those volumes were larger than the others (every other tube had a volume of 400 µL). The W0 samples were 700 µL
    • Added 70 µL of sodium acetate to W0 tubes, and 40 µL to all others (except for 31 WB, which I accidentally added 70 µL to instead of 40 µL)
  • Added 2 sample volumes of 100% ethanol (200 proof) to each sample
    • Added 800 µL to all sets except W0, which needed 1400 µL. The W0 tubes were extremely full, so I had to transfer each W0 tube to a corresponding 2.0 mL centrifuge tube. While I was doing this, Kaitlyn took care of sets WB and E2
  • Kaitlyn vortexed all sample centrifuge tubes while I put reagents away
  • Placed all tubes in a box, then placed box in the -20ºC freezer

Next Thursday, I’ll finish the precipitation process and quantify the reads. Almost done with labwork!

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Yaamini’s Notebook: Virginica MBDSeq Day 3

Dynabeads are dynamite

But first, a couple of things I forgot to mention I did yesterday:

  • Made 10 mL of the 1x Bind/Wash Buffer following the MethylMiner protocol
    • 2 mL 5x Bind/Wash Buffer mixed with 8 mL DNAse free water
  • Sample 106 had a very low concentration of DNA, so Sam set it in the speed vacuum to evaporate the liquid and further concentrate it before I used it

Today, I went through the protocol and 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: Calculate new concentration for sample 106

  • Sam removed the sample from the speed vacuum yesterday and placed the sample back in the fridge.
  • Used a pipet to measure volume of sample
  • Calculated the new sample concentration using the original sample concentration, the original volume, and the new volume
  • Calculated the volume of DNAse-free water needed to dilute the sample to 25 ng/µL, which is the concentration specified by the protocol
  • Added DNAse-free water to sample

Step 2: Prepare beads

  • Labelled ten 1.7 mL centrifuge tubes with sample number and “B” (ex. 31 B)
  • Obtained Dynabead stock from fridge
  • Resuspended beads by gently pipetting up and down
  • 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:
    • 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 a total of two times.

Step 3: Bind MBD-Biotin protein

  • Labelled ten 1.7 mL centrifuge tubes with sample number and “P” (ex. 31 P)
  • Obtained MBD-Biotin Protein from -80ºC freezer
  • 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

Step 4: 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 a total of four 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 5: Add DNA

  • Labelled ten 1.7 mL centrifuge tubes with sample number and “DNA”) (ex. 31 DNA)
  • Added 100 µL of 5X Bind/Wash Buffer to each tube
  • Added designated sample DNA volume from calcuations to each “DNA” tube
    • Here’s where I boofed a little. The first sample I added to its designated “DNA” tube was sample 106. I used all of the sample I had, since that’s what Sam and I discussed. I then decided to do the next three samples with the lowest concentrations: 108, 31, and 32. I added all of the sample volume from those tubes, since that’s what Sam and I discussed (normalizing our sample volumes to the ones with lowest concentrations). I went to pull 160 µL from the next sample, but there wasn’t enough sample volume! That’s when I realized that the rest of the samples had not been diluted to 25 ng/µL! I quickly used DNAse-free water to dilute the samples to the the appropriate concentration. For the DNA samples I had already into the “DNA” tubes, I diluted them in those tubes. I accidentally added the 60 µL of water I was going to use to dilute 108 DNA into 108 B. Seeing how the next steps involve me mixing everything anyways, I figured it should be okay. Just another case of me messing up in the best possible way.
  • Topped “DNA” tube volumes to 500 µL with DNAse-free water
  • Transfered “DNA” tube contents to corresponding “B” tubes
  • Mixed tubes on a rotating mixer overnight at 4ºC

One day of labwork done! Here’s what I need to do to first thing tomorrow:

  • Make 10 mL more of the 1X Bind/Wash Buffer
  • Label six sets of 1.7 mL centrifuge tubes for non-captured DNA, washes, and elutions

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