This experiment is part of the SCIENCE ENGINE Challenge Grant. Browse more projects

How will coldwater fish survive on a hot planet?

Michigan State University
East Lansing, Michigan
BiologyEcology
DOI: 10.18258/22782
Grant: SCIENCE ENGINE
$4,217
Raised of $4,215 Goal
100%
Funded on 2/03/22
Successfully Funded
  • $4,217
    pledged
  • 100%
    funded
  • Funded
    on 2/03/22

DNA Digestion and Extraction

Day 1: DNA Digestion

A standard proteinase k digestion approach was used to digest fin tissue samples for extraction. 

Steps:

  1. Pipette Lifton's buffer into each well of a 96 well plate. 
  2. Add a fin clip sample to each well (2mm in size) and decontaminate tools between samples. 
  3. Add digestion master mix ( Lifton's buffer, proteinase K, DTT) to each well. 
  4. Seal plate with foil, vortex, and incubate at 55 C overnight. 


Day 2: DNA Extraction

A standard bead extraction with ethanol wash was used to extract DNA from digested tissue. (This extraction protocol is designed for use with a Liquidator 96 Manual 96-well Pipettor from Rainin)

Steps: 

  1. Vortex to mix and spin down the plate to collect any condensation 
  2. To a new plate, add hybridization buffer (NaCl, PEG 8000, DTT) and AgenCourt AMPure XP beads, as well as the lysate 
  3. Mix and incubate for 5 minutes then place on a magnet 
  4. Aspirate the supernatant and discard
  5. Remove the plate from the magnet, add ethanol to re-suspend beads, mix, then place back on magnet, aspirate and discard 
  6. Repeat step 5
  7. Allow the beads to air dry while on magnet, then add a volume of low TE buffer to elute DNA from beads 


*This is a highly simplified version of the extraction protocol used. The full protocol, including reagent concentrations and volumes, can be found in the methods section of Ali et al 2016 under the subsection "Genomic DNA extractions"  (https://academic.oup.com/genet...)


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