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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
Mutant= 87 bp
Wild Type = 84 bp
Sequence
Wt Sequence (deletions in lower case):
GCTGTTATCTCCGCAGGTTTCGGCATGACAACACCAGCcacagtgggcgggaaggccttcctcatcgcctacggactgttcggctgcgcggggaccatcttgttcttcaacctctttctagagcgcatcatctcgctgctggccttcatcatgcgcgcctgccgggagcgccagctgcgccgcagtggcctgctgcccgccaccttccgccgcggctcggcgctgtcggaggccgacagcctggcgggctggaagccctcggtgtaccacgtgctgctcatcctgggcctgttcgccgtgctgctggcctgttgcgcctcggccatgtacaccagcgtggaaggctgggactacgtggactcgctctacttctgcttcgtcaccttcagcaccatcggcttcggggacctggtgagcagccaacacgccgcctaccggaaccaggggctctaccgcctgggcaacttcctcttcatcctgctcggcgtgtgctgcatctactcgctcttcaacgtcatctccatcctcatcaagcaggtgctcaactggatgctgcgcaagctgagctgccgctgctgcacgcgctgctgcccggctcccggcgcgcccctagcccggcgcaacgccatcaccccgggctcccggctgcgccgtcgcctggccgcgctgggtgccgaccccgcgactcgcgacagcgatgccgagggccgccgcctgtcgggcgagctcatctctatgcgcgacctcacggcgtccaacaaggtgtcgctggcgctgctgcagaagcagctgtccgagacggccaacggctacccgcgcagcgtgtgcgtcaacacgcgacagaacggcttctcgggcggcgtgggcgcgctgggcatcatgaacaaccggctggcagagaccagcgcCTCCAGGTAGAGCGGCCGCCCGCCCCCAGCGTCGCGGACCGGGCCGGGGCTGCGCGCCGCCGGGCGGGTTTGCTTCACTTCTCTCCGAGATGGCGCTTTCTTAA
This mutation is a 870 bp deletion beginning at Chromosome 17 position 87,745,950 bp and ending after 87,746,819 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 62729 | GTT GAG CAC CTG CTT GAT GA | Wild type Reverse | A | |||
| 67017 | AAC TTC CTC TTC ATC CTG CTC | Wild type Forward | A | |||
| 67018 | Fluorophore-1 | CGT GTG CTG CAT CTA CTC GCT | Quencher-1 | WT Probe | ||
| 67019 | CAC ACC ACA TGC GAC CAC | Mutant Forward | A | |||
| 67020 | GCT CTA CCT GGA GGC TGG T | Mutant Reverse | A | |||
| 67021 | Fluorophore-2 | CCT GGC TGT TAT CTC CGC AG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 62729 | 0.40 uM |
| 67017 | 0.40 uM |
| 67019 | 0.40 uM |
| 67020 | 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.