DNA Extraction of Adult Oyster Tissue - Part 4

~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 31, 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_3 M1
  • BAR_6 M1
  • BAR_10 M1
  • KIC_9 M1
  • MCD_7 M1
• Pull Proteinase k out of upright -20 freezer in Puritz Zymo Reagents box and put on ice
• Pull out Blue Solid Tissue Buffer and Nuclease-free water
• 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 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 mini centrifuge 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 600 rpm for 30 minutes
  • Halfway thru, spin down samples on mini centrifuge
  • Added 15 more minutes at 1000 rpm because samples were cloudy/mucousy
• 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 10 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 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 2 ul of DNA to the respective PCR tube for agarose gel
  • 1 ul will be taken directly from 1.5-ml tube for Qubit

7 ul remaining in all E1 1.5 ml tubes; 97 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	183 RFU
Std 2	22061 RFU
NAR_3 E1	290
NAR_3 E2	23.0
BAR_6 E1	129
BAR_6 E2	9.05
BAR_10 E1	78.0
BAR_10 E2	9.34
KIC_9 E1	146
KIC_9 E2	13.9
MCD_7 E1	77.5
MCD_7 E2	9.30

This time around, there’s a lot more DNA in both elutions, with the first elution having a ton of smaller fragments of DNA.

Agarose Gel Electrophoresis

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

• Only 2 ul of DNA for each sample were used
• Did not use diluted gelgreen this time
• Used a different loading dye - Purple loading dye versus Tritrack

Same as before, first elution has smaller fragments than second elution. Gel doesn’t look super pretty. I may run two or three of these samples on the TapeStation just to make sure everything is good. I also think I’ll run the gel longer next time.