DNA elution and ethanol precipitation (again)

Today I took the DNA samples bound to MBD-Biotin I placed on the rotating mixer yesterday, obtained the bound DNA, and started the ethanol precipitation process.

Step 0: Prepare for the day

• Labelled six sets of 10 1.7 mL centrifuge tubes. I made sure each set was a different color so it would be easier to tell the samples apart when working with many tubes.
  • Sample Number + Wash 0 (ex. 6 W0)
  • Sample Number + Wash A (ex. 6 WA)
  • Sample Number + Wash B (ex. 6 WB)
  • Sample Number + Elution 1 (ex. 6 E1)
  • Sample Number + Elution 2 (ex. 6 E2)
  • Sample Number + Elution 3 (ex. 6 E3)
• Got ice

Step 1: Remove non-captured DNA

• Removed tubes from the rotating mixer at 4ºC
  • I removed the samples at 8 a.m.
  • Batch 1 ran from 1 p.m.-8:30 a.m., and Batch 2 ran from 12:30 p.m.-8 a.m. Perfect timing!
• 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
  • 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

Step 3: Ethanol precipitation

• Placed non-captured DNA and wash tube (W0, WA, and WB) in the -20ºC freezer in FTR 213
• To each elution tube (E1, E2, and E3)
  • Added 1 µL glycogen (from -20ºC freezer)
  • Added 1/10th sample volume of pH 5.2 3 M sodium acetate (made by Sam in March 2007) to each sample
    • Sample volume was 400 µL, so I added 40 µL
  • Added 2 sample volumes of 100% ethanol (200 proof) to each sample
    • Added 800 µL to all tubes
• Gently mixed all tubes by pipetting gently
• Placed tubes in the -20ºC freezer
  • Batch 1 was in the freezer from 8/13 to 8/18. I will keep Batch 2 in the freezer from 8/19 to 8/24 to avoid any batch effects.

Going forward

1. Complete ethanol precipitation
2. Check methylated DNA concentrations
3. Send samples for sequencing