Using Zymo Quick-DNA Miniprep Plus Kit

Extracting DNA from juvenile oyster samples from multi-stressor exposure experiment.

Juveniles were collected individually in 1.5ml tubes, preserved with liquid nitrogen, and stored in -80 freezer until processing.

This is the general protocol I followed for DNA Extractions adapted from dual DNA/RNA extraction protcol. I typically processed 10-12 samples at a time.

Time to Completion: TBD hours (includes QC)

Prep tubes

Prep and label all tubes for extraction protocol (for each tube type, multiply by number of samples being processed)

tube	purpose	number
1.5 mL	homogenization/digest tube	* (# of samples)
1.5 mL	intmed. DNA steps	* (# of samples)
Yellow spin-column & collection tubes	DNA purificiation	* (# of samples)
1.5 mL	final DNA sample	* (# of samples)

Homogenization

• Turn on thermomixer and set to 55 degrees C, speed 1000 rpm
• Pull Proteinase K out of -20 freezer to warm to room temperature
• Pull Biotium dsDNA kit out of fridge/freezer and put in drawer (light sensitive) to warm to room temperature
• In homogenization/digest tubes, add 300 μL of DNA/RNA Shield
• Pull samples out of -80 freezer and put on ice
• Sterilize forceps with 100% EtOH, Type II DI Water, RNAse Zap, and RNAse-free Water
• Sterilize forceps between each sample
• Using forceps, transfer juvenile to 1.5 mL tube with DNA/RNA Shield
• Sterilize cone-shaped dremmel bit with 100% EtOH, Type II DI Water, RNAse Zap, and RNAse-free Water
• Sterilize the dremmel bit between each sample
• Insert dremmel bit into 1.5 mL tube with juvenile and grind up juvenile for ~10 seconds at speed 5
• Add 150 μL of Solid Tissue Buffer to each tube
• Add 10 μL of Proteinase K
• Vortex and spin down
• Put in thermomixer at 55 degrees C, shaking at 1000 rpm for 30 minutes
  • Halfway through, spin down in tabletop centrifuge at 13000 rpm for 1 minute

During incubation period:

• Prep 10 mM Tris HCl pH 8.0 for thermomixer
  • 50 μL x # of samples (plus error) in 1.5 mL tubes
  • Put in thermomixer set to 70 degrees C

After incubation:

• Spin down tubes at max speed for 2 minutes in tabletop centrifuge
• Without disturbing the debris pellet, gently transfer all supernatant (~450 μL) to new 1.5 mL tube (intmed. DNA step)
• Save debris pellet and store in -20 freezer (can check for incomplete digestion later on if necessary)

DNA Purification

• Add 400 μL of Genomic Binding Buffer to each intmed. DNA tube
• Vortex and spin down
• Add 800 μL of supernatant to yellow spin-column
• Centrifuge at 13000 rpm for 1 minute
• Discard flow-through
• Transfer spin-columns to new collection tube?????????????????????????
• Add 400 μL of DNA Pre-Wash Buffer to spin-column
• Centrifuge at 13000 rpm for 1 minute
• Discard flow-through
• Add 700 μL of g-DNA Wash Buffer
• Centrifuge at 13000 rpm for 1 minute
• Discard flow-through
• Add 200 μL of g-DNA Wash Buffer
• Centrifuge at 13000 rpm for 2 minutes; discard flow-through
• Transfer spin-column to final DNA 1.5 mL tube
• Carefully drip 50 μL of warmed 10 mM Tris HCl pH 8.0 directly onto filter
• Incubate at room temperature for 5 minutes
• Centrifuge at 16000 rpm for 30 seconds and discard spin column
• Should have 50 μL of DNA

Quality Control Check

Qubit

• DNA concentrations are checked using the Biotium dsDNA Broad Range quantitation kit
• use 1 μL of sample for each of the assays (unless you need to use more)
• Final DNA and RNA concentrations (ng/μL) can be accessed here

Agarose Gel

• DNA quality is checked using an Agarose Gel, following the protocol for a Small 1% gel run
• use 1uL DNA diluted in 4uL nuclease free water + 1uL loading dye for sample wells
• 100 V for 1 hr (60A)
• Sample gel can be accessed here