LDLR Genotyping
Modified from https://www.jax.org/Protocol?stockNumber=002207&protocolID=27075
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)
- 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
- 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.
| 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.