Single-Tube PCR Genotyping of Ptprca (JAXBoy, CD45.1) and Ptprcb (C57BL/6J, CD45.2)

Dillon Corvino

Validated allele-specific endpoint PCR using long-tailed primers.
Published

December 1, 2025

Protocol ID: MUS-PTPRC-GENO
Version: v1.1
Author: Dillon Corvino

Purpose

This protocol describes a validated single-tube endpoint PCR assay for distinguishing:

  • C57BL/6J (Ptprc^b/b, CD45.2)
  • JAXBoy (Ptprc^a/a, CD45.1)
  • Heterozygous (Ptprc^a/b)

The method is derived directly from Supplementary Tables S1, S4–S10 and Figures S1–S3 of Ryan et al. 2024.

Background

The Ptprc locus contains SNPs that differentiate the CD45.1 (Ptprc^a) and CD45.2 (Ptprc^b) alleles.

A single SNP assay using:

  • Ptprc_a_F3cT3 (long-tailed allele-specific forward primer)
  • Ptprc_b_F3d (allele-specific forward primer)
  • Ptprc_R3 (common reverse primer)

provides robust genotype resolution in one reaction tube.

Materials

Reagents

  • HotSHOT buffers
  • DreamTaq Green 2× Master Mix
  • Primers as detailed below
  • Nuclease-free water

Equipment

  • Thermocycler
  • 0.2 mL PCR tubes
  • Agarose gel system

Primer Sequences

Ptprc_b_F3d

TTG TTT GGA GTG GAA AAC GA

Ptprc_a_F3cT3
(81-nt tail + allele-specific region)

cat ctg ata ttt gaa aga ccc aag ccc tct cat acg cga cac gag atc tac act 
ctt tcc cta cac gac gct ctt ccg atc TGT TTG GAG TGG AAA ACC A

Ptprc_R3

CGT TGT GAA TTT GTT TCA GTG C

Ready-to-Use Primer Mix (Total 100 µL)

  • Ptprc_a_F3cT3 (100 µM): 13.3 µL
  • Ptprc_b_F3d (100 µM): 10 µL
  • Ptprc_R3 (100 µM): 10 µL
  • Water: 66.7 µL

Use 0.5 µL per 10 µL PCR reaction.

Genomic DNA Preparation (HotSHOT)

  1. Add 50–75 µL alkaline buffer to ear/tail biopsy.
  2. Heat 95°C × 30 min.
  3. Chill and neutralize.
  4. Spin briefly.
  5. Use 1–2 µL supernatant per PCR.

PCR Reaction Setup (10 µL total)

  • 2 µL HOTSHOT DNA
  • 5 µL 2× DreamTaq Green
  • 0.5 µL Primer Mix
  • 2.5 µL Water

PCR Cycling Conditions

Standard cycling (recommended):

  1. 94–95°C × 2 min
  2. 36 cycles of:
    • 95°C × 30 s
    • 56°C × 30 s
    • 72°C × 60 s
  3. 72°C × 5 min

Expected Amplicon Sizes

Genotype Size
Ptprc^b/b 243 bp
Ptprc^a/a 323 bp
Ptprc^a/b 243 + 323 bp

Gel Electrophoresis

  • 2–3% agarose
  • Run until 200–350 bp resolves
  • Load 5–10 µL PCR product

Controls

Include known:
- Ptprc^b/b
- Ptprc^a/a
- Ptprc^a/b
- NTC

Buffers Required

Full composition, preparation, storage, and safety information for each buffer lives in its own linked buffer document — only a concise per-buffer summary is reproduced here, generated automatically from that document.

None

Troubleshooting

  • Weak bands → increase template or cycles.
  • Allele dropout → check 56°C annealing temperature.
  • Non-specific bands → small increase in annealing temperature.

Version History

Version Date Description
v1.0 2025-12-01 Initial release.
v1.1 2026-09-18 Dillon Corvino - Replaced # Buffers used with an auto-generated # Buffers Required section (one heading + one small table per buffer, sourced from each buffer’s own summary: data), moved to the correct position after Procedure, and fixed leaked R package-startup warnings.

Citation

Ryan CE et al. (2024). Single-tube Ptprc SNP genotyping assay.