MBD-Biotin wash and binding (again) and ethanol precipitations

Similar to last week, I started the DNA and MBD-Biotin binding process with my five remaining samples (9, 48, 57, 59, and 63). These samples all required different input volumes since they did not have at least 4 µg of DNA, so I had to go even slower and refer to my spreadsheet with reagent volumes. Before I started, I prepared 10 mL of 1X Bind/Wash Buffer with 2 mL of 5X Bind/Wash Buffer and 8 mL of DNAse-free water.

Step 1: Prepare beads

• Labelled ten 1.7 mL centrifuge tubes with sample number and “B” (ex. 9 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. 9 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: 10:30 a.m., end: 11:30 a.m. (perfectly timed with lab meeting #efficiency)

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
• Obtained 5X Bind/Wash Buffer
• Diluted samples to 25 ng/µL with DNAse-free water
  • Sample 7 needed to be diluted with ~1700 µL of water so I needed to transfer the sample from the small shearing tube to a 2 mL centrifuge tube
• Labelled ten 1.7 mL centrifuge tubes with sample number and “DNA” (ex. 9 DNA)
• Added 100 µL of 5X Bind/Wash Buffer to each tube
  • Didn’t mess it up this time!
• Added designated sample DNA volume from calcuations to each “DNA” tube
  • Used all of samples 6 and 12
• 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
  • Placed the tubes in the mixer at 12:40 p.m.

After I added Batch 2 samples to the rotating mixer, I decided to finish the ethanol precipitation!

Step 0: Prepare for precipitations

• Set up centrifuge in the 4ºC frige in FTR 213
• Obtained samples from the -20ºC freezer in FTR 209 and placed them in the 4ºC fridge in FTR 213
• Made 70% EtOH with 7 mL 100% ethanol and 3 mL DNAse free water and placed it in the -20ºC freezer in FTR 209
• Labelled tubes to save ethanol precipitation supernatant
  • sample number and “E1 Sup” (ex. 6 E1 Sup)
  • sample number and “E2 Sup” (ex. 6 E2 Sup)
  • sample number and “E3 Sup” (ex. 6 E3 Sup)
  • sample number and “EtOH Sup” (ex. 6 EtOH Sup)

Step 1: Pellet DNA and consolidate

While I could pellet each E1, E2, and E3 tube separately and consolidate after resuspension, this allows me to increase my final DNA concentration and not work with so many tubes.

• For each “E1” tube
  • Centrifuged tubes for 15 minutes at 16,000 rcf at 4ºC
  • Pipetted supernatant from “E1” tube into corresponding “E1 Sup” tube without disturbing the pellet
  • Added “E2” tube contents into corresonding “E1” tube
• For each “E1” tube with “E2” supernatant
  • Centrifuged tubes for 15 minutes at 16,000 rcf at 4ºC
  • Pipetted supernatant from “E1” tube into corresponding “E2 Sup” tube without disturbing the pellet
  • Added “E3” tube contents into corresonding “E1” tube
• For each “E1” tube with “E3” supernatant
  • Centrifuged tubes for 15 minutes at 16,000 rcf at 4ºC
  • Pipetted supernatant from “E1” tube into corresponding “E3 Sup” tube without disturbing the pellet

Step 2: Ethanol precipitation

• Obtain 70% EtOH and place on ice
• For each “E1 tube”
  • Added 500 µL 70% ice cold EtOH into “E1” tubes
  • Centrifuged tubes for 5 minutes at 16,000 rcf at 4ºC
  • Pipetted supernatant from “E1” tube into corresponding “EtOH Sup” tube without disturbing the pellet
  • Repeated this entire process once more for a total of two times
• Air-dried the pellet fro 5 minutes
  • I avoided drying out the pellet fully as instructed by the protocol
• Resuspended DNA pellet with 25 µL Qiagen Buffer EB (method copied from Sam’s notebook) and placed samples in the -20ºC freezer in FTR 209

Going forward

1. Label six sets of 1.7 mL centrifuge tubes for non-captured DNA, washes, and elutions
2. Complete elutions and ethanol precipitations