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