Fetching SNPs

Contents

Fetching SNPs (Activities)

Exercises adapted from: Fetching SNPs: A Dog Genotyping Laboratory for Undergraduate Biology.

Lab 1: Collection/Isolation/Amplification

Sample Collection

Students who have access to canine companions  should have been provided a cytobrush and lysis buffer. Brush the inside of a dog’s mouth 15-20 times. Swirl the brush into the lysis buffer to dislodge cells and store the materials in the freezer until bringing to lab.

DNA isolation

  1. DNA in lysis buffer should be brought into lab on ice.
  2. Column purify genomic DNA from cells in lysis buffer as described by the kit available (protocol provided during class)
  3. Elute the DNA from column into collection tubes using H2O and store in freezer until use

PCR of SNP markers

  • 2X PCR Master Mix recipe for 30 μl reactions
Positive Control Rxn (μl) Genotyping Rxn (μl) Genotyping Rxn Master Mix for N dogs Component
3 3 – Genomic Template (10 ng/μl)
15 15 15(N+1*) Taq PCR Master Mix
– 1 1(N+1*) SNP Genotyping Primer Pair (10 μM)
1 – – SNP Positive Control Primer  Pair (10 μM)
11 11 11(N+1*) Water

* Ensure there is at least 10% extra Reaction Mix for pipetting error. For example, if the number of dogs, N, is between 10 and 20, multiply the component volumes by N+2 to ensure there is enough for all of your reactions. 

For each DS SNP marker:

  1. Mix together the ingredients for a Positive control reaction into a PCR tube.
    • Your instructor will tell you which dog sample to use for the template.
    • Label tube with DS# P, for example: “DS1-P”
    • Positive controls are essential as a reference for what the true digesting pattern should look like. These are known samples that will digest.
  2. Aliquot 3 μl of Dog DNA for each of your genotyping reactions into its own PCR tube, label with the Dog SNP number (DS#) and dog letter ID. For example “DS1-A”
  3. Add 27 μl of combined Genotyping Rxn Mix to each genotyping reaction. 

An example of a PCR setup for genotyping 3 dogs (A-C) with 2 SNP markers (DS1 and DS3):

Tube # SNP1 Tube # SNP3
1  DS1-PC 5 DS3-PC
2 DS1-A 6 DS3-A
3 DS1-B 7 DS3-B
4 DS1-C 8 DS3-C

After you put together the PCR reactions, set them on ice until the class is ready to place all of the reactions in the thermocycler.

The PCR program for the thermocycler is as follows:

Step 1 96°C    3’ Initial Denature
Step 2 94°C    30” Cycle Denature
Step 3 55°C    30” Anneal
Step 4 68°C   30” Extension/Elongation
Step 5 Go to Step 2 for 35 cycles Cycling
Step 6 68°C    3’ Final elongation
Step 7 4°C     ∞ Hold

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