DNA Sampling Protocol

DNA tissue samples should be taken using the protocols below and submitted to the Molecular Genetics Lab, Pacific Biological Station, Nanaimo. Samples should be submitted with proper labeling and documentation. In 2013 the Genetics Lab began the transition to Whatman paper for the collection of samples. Air drying tissue samples is an effective, cheap, safe, and environmentally friendly method of preserving DNA for molecular analysis. Contact Lab for a supplier of Whatman paper.

 Sample Sheets

  • A standard reporting sheet (Excel spreadsheet) should be filled out that accompanies the samples sent to the Molecular Genetics Lab, Pacific Biological Station, Nanaimo.
  • Minimum requirements on DNA sampling sheets:
    DATE, GEAR, AREA, SPECIES, SPECIMEN #, # TISSUES, SAMPLER,  CONTACT INFO
  • Additional information such as sub-area, location (lat-long), vessel name, statistical week, and comments may be needed in some circumstances.

Sample Labeling

  • Bulk or individual sampling should be given a unique alphanumeric code by collection agencies/organizations. For example, West coast of Vancouver Island Troll fisheries i.e. 01WCTDFO56
  • Format = Year + Fishery code + collection agency + vial number
  • Length = (2digits)+(3digits)+(3digits)+(>=1digit)

A. Whatman Sheet Sampling Instructions (preferred method)

  • Use punch to take a tissue sample from the preferred location on fish (see below).
  • The sampling procedure for the Whatman sheets involves taking a single sample from each fish at the thinnest part of the preferred tissue.
  • It is important to wipe down the tissue site on the fish prior to sampling.  This reduces cross-contamination caused by the slime between fish. It is also important to rinse the sampling tools between fish to avoid contamination.
  • The sample should be placed in the center of each of the squares printed on the paper.  The paper will absorb all the water in the tissue causing it to adhere to the paper.  If tissue too thick can be easily knocked off.
  • For the best results the tissue must be applied immediately after punching. (Short term storage of punches in water appears to be ok)
  • To prevent cross-contamination, try not to get too much liquid (slime) on the sheet or allow any liquid to run between squares.
  • Keep the sheet as dry as possible and avoid bending which might cause the samples to fall off.
  • Once the paper is completely dry it can be gently placed in the provided sleeves with the wax paper covering the samples.  This can then be placed in padded or stay-flat envelopes and shipped to our lab using regular mail.
  • Please ensure the date, species, fishing area and fishing gear (as well as any other pertinent information) is complete on the Whatman Sheet.
  • For video on the DNA sampling using Whatman paper: http://www.youtube.com/watch?v=W9opRMYVVqU

B. Scale Book DNA

  • DNA can be extracted and amplified from small amounts of tissue left on the scale where it attaches to the fish.
  • “Five down” sampling pattern in the scale book is preferred because at least two scales are needed for DNA extraction.
  • Scale books can be pressed for aging before scales are taken for DNA.
  • It is important to wipe down the scales on the fish prior to sampling.  This reduces cross-contamination caused by the slime between fish.
  • For further instruction on scale sampling: https://groups.nceas.ucsb.edu/monitoring-kb/dot/references/Guide-for-Sampling-Structures-used-in-Age.pdf

 C. Ethanol Sampling – individual or bulk sample

  • Use punch to take a tissue sample from the preferred location on fish (see below).
  • Bulk sample - where tissue from greater than one fish is placed in a bottle or vial, but only one tissue per fish. Individual sample - where tissue from one fish is placed in bottle or vial, there can be more than one tissue per fish.
  • Place tissue in sample bottle containing 95% non-denatured ethanol solution. Do not dilute the ethanol or use 70% ethanol. Do not use methanol or Reagent Alcohol solutions (i.e. rubbing alcohol or denatured alcohol) because these chemicals disrupt DNA amplification.
  • DNA will degrade if ratio of tissue to ethanol is too high. Should contain no more than ¼ tissue to ¾ ethanol.
  • Label each bottle with geographic location, statistical area, species, date and sampler.
  • If labels placed inside vials-- ***Do not use paper-based waterproof paper (i.e. Rite in the Rain©) because chemicals interfere with DNA extraction *** Plastic paper is ok.
  • Always label in pencil or solvent resistant markers to ensure the labeling does not get dissolved by the ethanol.

DNA Tissue sample in order of preference:

  1. Adipose punch- all salmon species - easiest for lab to process, sticks best to Whatman Sheets. Chinook and Coho salmon maybe missing adipose because used as external mark for coded wire tags.
  2. Caudal or other fin punch – sticks well to Whatman sheets. Rays can fall apart in bulk vials causing multiple amplification of same fish – works best for individual sample, good for non-lethal sampling.
  3. Scales – slower for the lab to process requires overnight extraction, sampling provides matched DNA and scale age.
  4. Operculum punch – can delaminate in bulk sample causing multiple amplification of same fish, works best for individual sample, can damage gills if live sampling, harder to process in the lab

Alternative tissues

  • Sometimes samples are previously frozen. Use the same sampling procedure as above except take small piece of muscle, heart, or liver instead. These tissues have more DNA so will amplify better after freezing. Do not add more the ¼ tissue to ¾ ethanol.
  • Axillary process this tissue is used by ADFG see website:
    http://www.genetics.cf.adfg.state.ak.us/ Non-lethal tissue sampling (PDF)

 

Diagram: tissue sample areas

Shipping Address:

Molecular Genetic Laboratory
Pacific Biological Station
3190 Hammond Bay Rd.
Nanaimo, BC V9T 6N7

For further information Contact:

John Candy 250-756-7224  or Liane Stenhouse - 250-756-7129