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