Sampling of 2 Pocilloporid Corals From the Aquarium Room, DNA Extraction, mtORF Amplification, and Sanger Sequencing Prep for Species Determination

Goal: Extract DNA from these two family Pocilloporidae corals and amplify them for the mtORF region, then prepare the amplifications for sequencing
Result: Good yields from extraction, and good yields from amplification
Major takeaways: Clipping directly into DNA/RNA shield was a good idea, I should have done a gel of the extraction even though I was time limited because that is good practice

Sampling and DNA Extraction

Using the Zymo Quick-DNA Miniprep Plus kit and glass bead tubes on two samples. Sample 1 is a Pocillopora coral and Sample 2 is a Stylophora coral.

• Made 2 1.5mL tubes, and used 1 bead tube to transfer half the beads into each 1.5mL tube
• Added 500ul of DNA/RNA shield into each tube
• Sterilzed clippers with 10% bleach, DI, and 70% etoh (before and between corals)
• Clipped chunk of coral directly into bead tube and replaced back into the tank in the AQ room
• Vortexed tubes for 1 minutes at max speed
• Spun down tubes
• Added 250ul of blue solid tissue buffer
• Added 16.6ul proteinase K
• Vortexed and spun down tubes
• Incubated tubes in the thermomixer for 1 hour at 55 degrees C, shaking at 900rpm
• Put a 1.5mL tube with 10mM Tris HCl in the thermomixer to warm up to 55 degrees C
• Spun down tubes
• Transferred liquid to new 1.5mL tubes
• Centrifuged tubes for 1 minute at 12,000rcf to pellet debris
• Transferred 600ul of supernatant to new 1.5mL tubes
• Added 1 volume (600ul) genomic binding buffer to each tube
• Vortexed and spun down tubes
• Added 700ul to yellow spin columns
• Centrifuged at 16,000rcf for 1 minute
• Discarded flowthrough into Zymo kit waste
• Added the remaining volume from the tubes into the columns
• Centrifuged at 16,000rcf for 1 minute
• Discarded flowthrough into Zymo kit waste
• Transferred the columns to new collection tubes
• Added 400ul of DNA pre-wash buffer to each column
• Centrifuged at 16,000rcf for 1 minute
• Discarded flowthrough into Zymo kit waste
• Added 700ul of DNA wash buffer to each column
• Centrifuged at 16,000rcf for 1 minute
• Discarded flowthrough into Zymo kit waste
• Added 200ul of DNA wash buffer to each column
• Centrifuged at 16,000rcf for 1 minute
• Transferred columns to new 1.5mL tubes (labeled for final tubes)
• Discarded flowthrough into Zymo kit waste
• Added 50ul warmed 10mM Tris HCl to each column
• Incubated 5 minutes at RT
• Centrifuged at 16,000rcf for 1 minute
• Added 50ul warmed 10mM Tris HCl to each column
• Incubated 5 minutes at RT
• Centrifuged at 16,000rcf for 1 minute
• Placed final tubes on ice

Qubit

• Broad Range dsDNA Qubit protocol

Sample	DNA Standard 1 (RFU)	DNA Standard 2 (RFU)	DNA 1 (ng/µl)	DNA 2 (ng/µl)	Average DNA (ng/ul)
1	178	19033	39.8	39.4	39.6
2	-	-	25.4	25	25.3

mtORF Amplification Cleanup and Sequencing Prep

Followed the mtORF Amplification Protocol exactly, except a TapeStation was run on the samples after cleanup instead of a gel.
Briefly:

• Diluted samples to 10ng/ul in 10ul:
  • 1: 2.525ul of DNA and 7.475ul 10mM Tris HCl
  • 1. 3.97ul of DNA and 6.03ul 10mM Tris HCl
• Master mix for triplicate reactions + one control:
  • 150ul phusion amplification mix
  • 3.9ul of FatP6 primer (10uM)
  • 3.9ul of RORF primer (10uM)
  • 132ul ultra pure water
• Aliquoted 97ul into 1 tube for each sample and 32ul into one tube for the control
• Added 3ul of diluted DNA into respective 97ul tubes
• Vortexed and spun down
• Split up 100ul tubes into 6 33ul tubes
• Added 1ul ultra pure water to the control tube
• Spun down tubes
• Placed in the thermocyler FATP RORF program
• Afterwards, combined triplicate samples and added 100ul of KAPA Pure Beads for a 1X clean up, 33ul for the control
• Performed normal cleanup
• Eluted in 50ul of ultra pure water
• Qubit of amplified DNA (protocol):

Sample	DNA Standard 1 (RFU)	DNA Standard 2 (RFU)	DNA 1 (ng/µl)	DNA 2 (ng/µl)	Average DNA (ng/ul)
1	179	15906	67.8	67	67.4
2	-	-	61.8	60.8	61.2
control	-	-	too low	-	-

• D5000 TapeStation to check for bands at right size (~1000 bp)
  • Results
• 4ul of 3.2mM RORF primer is needed
  • 1.28ul of 10mM primer
  • 2.72ul of ultra pure water
• 1:5 dilution of amplified DNA from each sample
  • 2ul of DNA
  • 8ul of ultra pure water
• New concentrations for each sample:
  • 1: 13.48ng/ul
  • 2: 12.26ng/ul
• Amount of DNA and water for 10ul containing 25ng of DNA:
  • 1: 1.85ul of diluted DNA and 8.15ul of ultra pure water
  • 2: 2.03ul of diluted DNA and 7.97ul of ultra pure water
• Added 2ul of the 3.2mM RORF primer to each tube
• Spreadsheet for GSC:

Sample IDa	Well (GSC use only)	Template Typeb	A. Template Size (bases)	B. Template Stock Conc. (ng/µl)	C. PCR template: ng needed = ((A ÷ 100) x 1.25) x 2	D. PCR template: Volume = (C ÷ B) µl	F. Volume PCR-H20 needed (10 minus D or E) µl	G. Volume primer needed 1 µl per reaction
HAQ1		PCR	1000	13.48	25	1.85	8.15	2
HAQ2		PCR	1000	12.26	25	2.03	7.97	2

• Samples will be brought up for sequencing on Monday and should be sequenced on Tuesday