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. Look on the tube from IDT, for example if there is 23.7 nmoles then you would resuspend in 237 µL of water
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.