Finishing ethanol precipitations

Today I completed the ethanol precipitations I started previously.

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. 9 E1 Sup)
  • sample number and “E2 Sup” (ex. 9 E2 Sup)
  • sample number and “E3 Sup” (ex. 9 E3 Sup)
  • sample number and “EtOH Sup” (ex. 9 EtOH Sup)

Step 1: Pellet DNA and consolidate

• 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 for 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. Measure concentration of precipitated DNA
2. Send samples for sequencing