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Notes
Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
The genotyping protocol(s) presented here have been optimized for reagents and conditions used by The Jackson Laboratory (JAX).
To genotype animals, JAX recommends researchers validate the assay independently upon receipt of animals into their facility.
Reaction cycling temperature and times may require additional optimization based on the specific genotyping reagents used.
Expected Results
>chr3:126438013+126438116 104bp CGGGACTTCAGCATTCTATCA ATGGGTTGAATAGCCGACCT
Mut = 119 bp
Wt = 104 bp
Fam = Mut
Hex = Wt
Sequence
Wt Sequence (deletions in lower case):
CAGATACACACGGGACTTCAGCATTCTATCATTAGACCTACCCAAcccaactgtctacctctggacaatgcaaccctacctcagaagctaaaggaggtcggctattcaacccatatggttgggaagtggcacttgggattttacagaaaagactgcatgcccaccaagagaggatttgataccttttttggctctcttttgggaagtggtgattattatacacactacaaatgtgacagtcctggggtgtgtggctatgacttgtacgaaaacgacaatgcagcttgggactatgacaatggcatctattcaacacagatgtacacgcagagagtgcagcaaatcttagccacccatgatcccacaaaacctctatttttatatgtggcttaccaagctgtccactcaccacttcaagcccctggcaggtactttgaacactacagatccattatcaacataaacaggaggaggtacgcagccatgctctcctgcttggatgaagcaatccacaatgtgaccctggccctaaagaggtatggtttctataacaatagcattatcatttactcctcagataatggtgggcagcccacagcaggaggaagtaactggccactcagaggcagcaaaggaacatattgggaaggaggcatccgggcagttggctttgtgcatagcccacttctaaaaaacaagggaacggtatgtaaggaacttgtgcacatcaccgattggtacccaaccttgatttcactggctgaaggacagattgatgaagacattcagctagatggatacgatatctgggagaccataagcgaaggtcttcgttcaccccgagtggatattttgcacaacattgaccccatttacaccaaggcgaaaaatggctcctgggcagcaggctatgggatctggaacactgcaatccagtcggccatccgggtacaacactggaaactgctcacaggcaatcctggctacagtgactgggtcccccctcaggctttcagcaatctgggcccaaaccggtggcacaatgaaaggattaccttgtcaactggcaagagtatctggcttttcaacatcacggctgatccgtatgagagggtggacctatccagcaggtatccaggcatcgtgaagaagctgctgcggagactgtcacaattcaacaagactgccgtgcctgtcaggtaccccccgaaggatcccagaagcaaccctaggctcaacggaggagtctggggtccgtggtataaagaggaaaacaagaaaaagaagtcaaacaaaaccaaggctaagaaaatgcaaaagaaaaaaagtaaggcaaggatgaggaagcagttggcagctcactcctcaataaaatgccacccaagtgttgccactggctaaaTGCAAAACTTTCTGCCTTGGTTAAACTTAGACTGCCTGGTTTCTAGGTAATTCAAAGAAATTGGCTCAGAAATACCTCGGCCAACCGCCTCGGGCTTG
This mutation is a 1357 bp deletion beginning at Chromosome 3 position 126,438,048 bp and ending after 126,439,404 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 52402 | CGG GAC TTC AGC ATT CTA TCA | Common | A | |||
| 52403 | ATG GGT TGA ATA GCC GAC CT | Wild type Reverse | A | |||
| 52404 | TTG GCC GAG GTA TTT CTG AG | Mutant Reverse | A | |||
| 52405 | Fluorophore-1 | ACC TCT GGA CAA TGC AAC CC | Quencher-1 | WT Probe | ||
| 52406 | Fluorophore-2 | CCA ATG CAA AAC TTT CTG CCT T | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 52402 | 0.40 uM |
| 52403 | 0.40 uM |
| 52404 | 0.40 uM |
| Wt Probe | 0.15 uM |
| Mutant Probe | 0.15 uM |
| DNA |
Cycling
| Step | Temp °C | Time | Note |
|---|---|---|---|
| 1 | 95.0 | -- | |
| 2 | 95.0 | -- | |
| 3 | 60.0 | -- | |
| 4 | -- | repeat steps 2-3 for 40 cycles | |
| 5 | 40.0 | -- | Forever |
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.
Strains Using This Protocol
This is the only strain that uses this protocol.