DNA Extraction of Adult Oyster Tissue - Part 3

~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 25, 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_5
  • NAR_8 - mantle and gill
  • KIC_3
  • MCD_2
  • MCD_5
• Pull Proteinase k out of upright -20 freezer in Puritz Zymo Reagents box and put on ice
• 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 labeling new 1.5-ml tubes
  • Ended up being closer to 15-20 minutes
• Pull out Blue Solid Tissue Buffer from Zymo Research Quick DNA Miniprep kit
• In newly labeled 1.5-ml tubes, add 95 ul of nuclease-free water, 95 ul of solid tissue buffer, and 10 ul of Proteinase k
  • Vortex and spin down in mini centrifuge
• Sterilize forceps with 10% bleach, Type II DI water, and 70% EtOH
• Transfer tissue pieces from PBS tube to respective 1.5-ml tube
  • Sterilize forceps before each sample
• Vortex samples for 10 seconds and spin down in mini centrifuge
• Put in thermomixer at 55 deg C at 600 rpm for 45 minutes
  • Using half speed from what was done with previous samples
  • Tissue was not fully solubilized after 45 minutes, so increased speed to 1000 rpm for 30 more minutes
• Once tissue is fully solubilized, place all samples in tabletop centrifuge and spin at 13000 rcf for 2 minutes
  • 1 minute longer than previous samples
• Make new set of labeled 1.5-ml tubes
• Pipette all supernatant (200 ul) to labeled 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 (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 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 50 ul of warmed 10 mM Tris HCl pH 8 to each spin column by dripping directly over the filter without touching it
• 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
• Transfer spin columns to second set of labeled 1.5-ml tubes, add 50 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 5 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

47 ul remaining in each 1.5 ml sample tubes

Qubit dsDNA BR assay

The captured pools were quantified following Qubit protocol for BR DNA

Sample	Avg ng/μl
Std 1	174 RFU
Std 2	19572 RFU
NAR_5 E1	45.5
NAR_5 E2	6.51
NAR_8 M E1	72.3
NAR_8 M E2	9.29
NAR_8 G E1	60.6
NAR_8 G E2	8.55
KIC_3 E1	30.8
KIC_3 E2	3.45
MCD_2 E1	35.5
MCD_2 E2	3.41
MCD_5 E1	30.4
MCD_5 E2	2.97

Much smaller quantities of DNA in the second elution, these have the larger fragments.

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

The second elution of each sample has the high quality DNA but in low quantity.

Plan for next set of extractions:

• Do PBS soak again
• Maybe dissect more gill tissue
• When separating the two elutions, use a smaller volume for elution 1 and larger volume for elution 2(?)