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
- 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