DNA Extraction of Adult Oyster Tissue - Part 9

~10 wild adult oysters were collected from 5 populations in Narragansett Bay

• Narrow River
• Green Hill Pond
• Barrington River
• Kickemuit
• Mary C. Donovan Marsh

Mantle and gill tissue were dissected from the oysters. Dissection protocol can be accessed here.

DNA Extractions

Completed on February 23, 2021

Zymo Research Quick-DNA Miniprep Plus used for DNA extractions of adult oyster tissue

• Pull samples out of -80 freezer and put on ice
  • NAR_6 M1
  • GHP_4 M1
  • BAR_2 M1
  • BAR_4 M1
  • MCD_3 M1
• Pull Proteinase k out of upright -20 freezer in Puritz Zymo Reagents box and put on ice
• Pull out Type II DI water, 10X PBS solution, Blue solid tissue buffer, and Nuclease-free water
• In original sample tube, add 90 ul of Type II DI water and 10 ul of 10X PBS (Phosphate Buffered Saline) solution to create 1X PBS solution soak
  • Vortex and spin down in mini centrifuge
• Let soak for 5-10 minutes while preparing next set of tubes
• In 2 ml Homogenization Tubes with ceramic beads, add 190 ul of Blue Solid Tissue Buffer and 190 ul of Nuclease-free water
  • Vortex and spin down
• Sterilize forceps with 10% Bleach, Type II DI Water, and 70% EtOH
• Use forceps to transfer tissue piece from original tube with PBS solution to 2 ml Homogenization tube with ceramic beads at solid tissue buffer and nuclease-free water
  • Sterilize forceps before each sample
• Setup tissuelyser II
  • Make sure to balance both tube racks
• Homogenize tissue samples in tissuelyser II for 2 minutes at 30 Hz
  • This will create a ton of bubbles in the sample tubes
• Let the samples sit for about 3 minutes then spin down on tabletop centrifuge at 13000 rcf for 1 min to bring down bubbles
• Add 20 ul of Proteinase k to each sample
  • Vortex for 10 seconds then spin down on mini Centrifuge
• Put samples in thermomixer at 55 deg C at 1000 rpm for 30 minutes
  • Halfway thru, spin down samples on tabletop centrifuge at 13000 rcf for 1 min
• Make new set of labeled 1.5 ml tubes
• After 30 minute incubation, place all samples in tabletop centrifuge and spin at 13000 rcf for 2 minutes
• Transfer all supernatant (400 ul) to the newly labeled 1.5 ml tubes
• Make 1.5 ml tube of 10 mM Tris HCl pH 8 and place in thermomixer at 70 deg C
• Set up tubes for extraction - 1 yellow spin column inside a collection tube for each sample - label lid of spin column
• Get liquid waste beaker from sink near -80 freezers
• Add 2 parts volume (800 ul) of Genomic Binding buffer to each tube
• Vortex for 5 seconds and spin down in mini centrifuge
• Add 800 ul of sample to their labeled yellow spin column
• Centrifuge spin columns at 13000 rcf for 1 minute
• Pour off flow through in liquid waste beaker
• Put spin columns in same collection tubes
• Add remaining liquid (400 ul) from each sample to labeled yellow spin column
• Centrifuge spin columns at 13000 rcf for 1 minute
• Pour off flow through in liquid waste beaker
• Transfer spin columns to new collection tubes and discard of old collection tubes
• Add 400 ul of DNA pre-wash buffer to each spin column
• Centrifuge at 13000 rcf from 1 minute
• Pour off flow through in liquid waste beaker
• Place spin columns in same collection tubes
• Add 700 ul of g-DNA wash buffer to each spin column
• Centrifuge spin columns at 13000 rcf for 1 minute
• Pour off flow through in liquid waste beaker
• Put spin columns in same collection tubes
• Add 200 ul of g-DNA wash buffer to each spin column
• Centrifuge spin columns at 13000 rcf for 1 minute
• Make final 1.5 ml tubes - label lid with sample id, elution #, and DNA; label side with initials, date of extraction, sample id, elution #, DNA, and C. virginica
  • Make 2 tubes for each sample - splitting the two elutions into two separate tubes
• Transfer spin columns to first set of labeled 1.5 ml tubes
• Pour off flow through in liquid waste beaker and discard collection tubes
• Take warmed 10 mM Tris HCl pH 8 out of thermomixer
• Add 30 ul of warmed 10 mM Tris HCl pH 8 to each spin column by dripping directly over the filter without touching it
• Immediately place tubes in centrifuge with all the lids of the 1.5 ml tubes facing the same direction and centrifuge at MAX speed for 1 minute
  • No incubation period for the first elution
• Take tubes out - DO NOT pour off liquid, keep in tube
• Transfer spin columns to second set of labeled 1.5-ml tubes, add 100 ul of warmed 10 mM Tris HCl pH 8 to each spin column by dripping directly over filter without touching it
  • Put first set of 1.5-ml tubes with the first elution on ice
• Incubate for 10-15 minutes
• Centrifuge at max speed for 1 minute
• Take out spin columns at discard
• Make labeled PCR strip tubes - 1 for each elution = 2 per sample
  • Add 1 ul of DNA and 4 ul of nuclease-free water to the respective PCR tube for agarose gel
  • 1 ul will be taken directly from 1.5-ml tube for Qubit

28 ul remaining in all E1 1.5 ml tubes; 98 ul remaining in all E2 1.5 ml tubes

Qubit dsDNA BR assay

The captured pools were quantified following Qubit protocol for BR DNA

Sample	Avg ng/μl
Std 1	182 RFU
Std 2	17996 RFU
NAR_6 E1	123
NAR_6 E2	6.88
GHP_4 E1	113
GHP_4 E2	5.88
BAR_2 E1	172
BAR_2 E2	12.1
BAR_4 E1	105
BAR_4 E2	6.39
MCD_3 E1	84.8
MCD_3 E2	7.07

Smaller amount of DNA in the E2 elutions.

Agarose Gel Electrophoresis

The DNA quality and size were assessed following Agarose Gel Protocol for a small 1% gel.

• Only 1 ul of DNA for each sample were used
  • 4 ul of nuclease-free water added to bring volume up to 5 ul
• Did not use diluted gelgreen this time
• Used a different loading dye - Purple loading dye

Overall, the DNA looks really good.