Genotyping mice from ear clips
Elizabeth Phelan
Abstract
This is a protocol that describes the genotyping procedure from ear clip samples. This includes a general PCR protocol for primers with annealing temperatures in the range 55-70 degrees C. For other primers, the thermal cycling should be adjusted.
Steps
Tissue Collection
Scruffing mouse securely, snip a tissue sample approximately 4mm2 in size from the edge of its ear using a punch or scissors.
Store and transport samples separately in clearly-labelled 1.5mL tubes.
DNA Extraction
Add 200uL of 20mM NaOH to each tissue sample.
Heat samples to 100 degrees C for 15 minutes, or until tissues are thoroughly dissolved.
Add 50uL of 30% TRIS to each sample and centrifuge on high speed for 6 minutes.
Withdraw 200uL of supernatant from each sample, store in 1mL tube. Use 1uL of this DNA solution for each reaction.
PCR amplification
Create master mix. Scale depending on how many reactions you are conducting.
To create the mix, for each reaction add in order:
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17 uL of autoclaved water
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5 uL of GoTAQ buffer
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0.5uL of dNTP
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0.5uL of each primer
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0.5uL of TAQ
Shake gently and add 24uL of this solution to DNA in a PCR plate.
Thermal cycle; the PCR protocol will vary according to primers, but cycling between 60 and 94 degrees Celsius has been effective for our lab.
Gel electrophoresis
Prepare gel:
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Heat 3g of agarose in 200mL TAE buffer solution.
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Once cooled to 55 degrees C, add 12uL SYBR Safe DNA Gel Stain
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Pour the gel onto the mold, add combs, and allow it to solidify for 15 minutes.
Place solid gel into the electrophoresis chamber. Load PCR product from the plate into the wells, one per well, leaving a gap between each well. Add a ladder before the first and after the last well, or as desired.
Switch on the electrical current (around 90 volts is recommended). Run the gel for 15-30 minutes depending on desired band size.
Remove the gel and image. Using an Anxygen gel reader is recommended.
