DNA Extraction of Adult Oyster Tissue - Part 1

~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 January 18, 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_7 M2
  • BAR_5 M2
  • BAR_7 M2
  • GHP_8 M2
  • KIC_6 M1
• Pull Proteinase k out of upright -20 freezer in Puritz Zymo Reagents box and put on ice
• Pull out Blue Solid Tissue Buffer from Zymo Research Quick DNA Miniprep kit
• To each original tube with the tissue piece, add 95 ul of nuclease-free water, 95 ul of solid tissue buffer, and 10 ul of Proteinase k
  • Vortex for 10 seconds and spin down on mini centrifuge
• Place tubes in thermomixer at 55 deg C shaking at 1200 rpm
  • Check tissue after 30 minutes
    • The liquid was pretty cloudy, so I added another 15 minutes and then another 30 minutes
  • After 1 hour and 15 minutes, the liquid still looked cloudy so I decided to move forward with the dissection protocol

NAR_7 M2: BAR_5 M2: BAR_7 M2: GHP_8 M2: KIC_6 M1:

• After incubation, place all samples in the tabletop centrifuge and spin at 13000 rcf for 1 minute
• Make new set of labeled 1.5 ml tubes
• Pipette all supernatant (200 ul) to each new tube
• 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 (400 ul) of Genomic Binding buffer to each tube
• Vortex for 5 seconds and spin down in mini centrifuge
• Add 400 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 (200 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 lide with sample id and DNA; label side with initials, date of extraction, sample id, DNA, and C. virginica
• Transfer spin columns to 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 50 ul of warmed 10 mM Tris HCl pH 8 to each spin column by dripping directly over the filter without touching it
  • Added too much Tris to KIC_6, so I’ll add ~25 ul of Tris during the second elution for this sample
• Incubate for 5 minutes
• 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
• Take tubes out - DO NOT pour off liquid, keep in tube
• Add an additional 50 ul of warmed 10 mM Tris HCl pH 8 to each spin column by dripping directly over filter without touching it
• 100 ul of DNA total
• Only 25 ul of Tris added to KIC_6 for this elution
• Incubate for 5 minutes
• Centrifuge at max speed for 1 minute
• Take out spin columns at discard
• Aliquot 6 ul of each sample to their respective PCR strip tube
  • 3 ul for agarose gel
  • 1 ul for Qubit
  • 1 ul for TapeStation (if needed)
  • 1 ul for error

94 ul remaining in 1.5 ml sample tubes

_Qubit dsDNA BR assay__

The captured pools were quantified following Qubit protocol for BR DNA

• 199 ul x 7.2 samples = 1432.8 ul of Buffer
• 1 ul x 7.2 samples = 7.2 ul of reagent

Sample	Avg ng/μl
Std 1	179 RFU
Std 2	18232 RFU
NAR_7	39.6
BAR_5	30.6
BAR_7	50.2
GHP_8	43.9
KIC_6	17.2

Agarose Gel Electrophoresis

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

• Only 3 ul of DNA for each sample were used
• Used a diluted gelgreen made by Maggie Schedl

The samples still look a bit streaky, so I’ll run them on a TapeStation.

TapeStation

Completed on January 20, 2021

Samples were run on the TapeStation following the protocol for Genomic DNA.

• Ran samples BAR_5 and KIC_6

See full report here.

KIC_6 looked really good, BAR_5 had a lot of smaller fragments around 1500 bp. Next time I will try smaller pieces of tissue.