mtORF Amplification of HMW P. acuta

Amplifying mtORF region from HMW Pocillopora Extraction and Sequencing Prep

Goal: Use extracted HMW DNA to amplify mtORF region in coral to confirm species
Result: Good yields from amplification
Major takeaways: Even though I had to dilute a lot for 10ng/ul, there were no issues

Sample Info Used coral P2185 from HoloInt Project, tube 3 from this HMW extraction

mtORF Amplification, Cleanup and Sequencing Prep

Followed the mtORF Amplification Protocol exactly. Briefly:

  • Diluted samples to 10ng/ul in 10ul:
    • 2ul of DNA and 38.75ul TE buffer
  • Master mix for 1 triplicate reaction:
    • 50ul phusion amplification mix
    • 1.3ul of FatP6 primer (10uM)
    • 1.3ul of RORF primer (10uM)
    • 44ul ultra pure water
  • Aliquoted 97ul into 1 tube
  • Added 3ul of diluted DNA into the 97ul tube
  • Vortexed and spun down
  • Split up 100ul tubes into 3 33ul tubes
  • 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
  • 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)
P2185 189 20669 61 61 61
  • 1% gel run at 100V for 60 minutes, band size is expected to be 1000bp:
  • 2ul of 3.2mM FATP6.1 primer is needed, 5ul was made
    • 1.6ul of 10mM primer
    • 3.4ul of ultra pure water
  • 1:10 dilution of amplified DNA
    • 2ul of DNA
    • 18ul of ultra pure water
  • New concentration should be:
    • 6.1ng/ul
  • Amount of DNA and water for 10ul containing 25ng of DNA:
    • 4.1ul of diluted DNA and 5.9ul of ultra pure water
  • Added 2ul of the 3.2mM FATP6.1 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
HAQ3   PCR 1000 6.1 25 4.1 5.9 2
  • Brought up to GSC 20210222
Written on February 22, 2021