DNA Extraction quick n' dirty (beginning steps)

Last week I shadowed Hollie while she performed the first few steps of DNA extraction. Here’s what I learned about the process and general tips for working in that lab:

  • Wear protective clothes at all time
  • Leave a “dirty” notebook/pen set in the lab
    • Need to get a lab coat -> can purchase things at the Chem. building’s stock room; need to use UW account and a project #.
  • Extracting process = ~2hrs
  • Extracting + quant. & qual. analysis = all day

Preparing workspace/materials:

  • Turn on heat block
  • Get dry ice in small styrofoam cooler
  • Wipe down station & all materials with “ELIMINase” -> this cleans enzymes/DNA from station
  • Prep vials:
    • Centrifuge can fit up to 24 vials, so Hollie processes 24max/day
    • Pour vials onto clean workspace, close caps, set into tray
    • Select samples; randomize selection at each step to avoid batch effects.
    • Label vials
  • Prep scoops/tool that will be used to measure sample amount; in this case Hollie had homogenized the geoduck into a frozen powder.
    • Soak/sterilize in ELIMINase
    • Rinse in DEPC/DI H20
    • Hold scoops/tool in sterilized falcon tube on dry ice

Work from DNeasy Blood & Tissue Kit

  • Check dates on kit and if ethanol has been added to chems.
  • Pipette in Proteinase K chemical (pretty viscous, so do this one first); 180 ul/<25mg tissue
  • Vortex then spin briefly to get chem to bottom of vial
  • Add 180 ul ATL Buffer (not touching vial with pipette tip) -> this lyses the cells

Transfer sample to vials

  • Take dry ice to -80 freezer; pull desired samples and put directly onto dry ice. Minimize time out of freezer/off ice: seconds matter for RNA; minutes matter for DNA
    • It’s important to organize tubes when storing to make pulling desired sample easy
  • Put ~25mg of sample into vials using prepared scoops/tools
  • Incubate at 56degC for ~1hr; should turn from clear to ~greenish (for geoduck samples)
  • Add 200ul AL buffer to all samples; vortex to mix
    • Incubate for ~10mins to avoid precipitation
  • Label columns
  • Transfer incubated samples into columns using pipette
    • Hopefully all of the sample volume fits into the column at once; if not, then may need to centrifuge, then add the rest.
  • Centrifuge @ 6000 for 1min
  • Add 500 ul AW1; centrifuge & discard waste & waste-catching outer tube
  • Add 500ul AW2; centrifuge & discard waste & tube

At this point the DNA is affixed to the column.

This is where I had to leave; will shadow again for remainder of steps

Written on October 10, 2016