LDLR Genotyping

Modified from https://www.jax.org/Protocol?stockNumber=002207&protocolID=27075

• SOP - Ethidium Bromide
• SOP - Electrophoresis

Reagents Needed

PCR Primers

• Ldlr Common Forward: 5'-TAT GCA TCC CCA GTC TTT GG-3'
• Ldlr Wild-type Reverse: 5'-CTA CCC AAC CAG CCC CTT AC-3'
• Ldlr Mutant Reverse: 5'-ATA GAT TCG CCC TTG TGT CC-3'

Master Mix

• DreamTaq Green PCR Master Mix (2X) - Thermo Fisher Scientific, Cat# K1081

Additional Reagents

• Nuclease-free water
• Template DNA (tail lysate or purified genomic DNA)

Primer Preparation

Stock Primer Preparation (100 µM)

1. Resuspend each lyophilized primer in nuclease-free water to 100 µM concentration
2. Store at -20°C

Primer Master Mix (10 µM each primer)

For a 1 mL primer master mix:

• 100 µL of 100 µM Primer 19799
• 100 µL of 100 µM Primer 19800
• 100 µL of 100 µM Primer oIMR7770
• 700 µL nuclease-free water

Final concentrations in primer master mix: 10 µM each primer

Store at -20°C

PCR Master Mix Preparation

Per Reaction (25 µL total volume)

• 12.5 µL DreamTaq Green PCR Master Mix (2X)
• 3.75 µL Primer Master Mix (10 µM each)
• 7.75 µL nuclease-free water
• 1 µL template DNA

Final primer concentrations in PCR: 1.5 µM each primer

For Multiple Reactions

Prepare master mix for (n+1) reactions, where n = number of samples:

• DreamTaq Green PCR Master Mix (2X): 12.5 µL × (n+1)
• Primer Master Mix: 3.75 µL × (n+1)
• Nuclease-free water: 7.75 µL × (n+1)

Aliquot 24 µL of master mix per tube, then add 1 µL template DNA to each

PCR Cycling Program

This program takes 1:50 to run.

Ldlr PCR Program

Step	Temperature (°C)	Time	Cycles	Notes
Initial Denaturation	94	3 min	1
Touchdown Phase
Denature	94	30 sec	10	Decrease annealing temp by 0.5°C per cycle
Anneal	65→60	30 sec
Extend	68	45 sec
Standard Cycles
Denature	94	30 sec	28
Anneal	60	30 sec
Extend	72	45 sec
Final Extension	72	5 min	1
Hold	4	∞

Expected Results

• Wild type: 351 bp
• Heterozygote: 179 bp AND 351 bp
• Mutant: 179 bp (GC-rich band)

Note: Run on 2% agarose gel. The mutant band (179 bp) is GC-rich and may appear fainter than expected.