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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
>chr4:117887454-117887547 94bp GGATCTGCAGTGATTGGTGA CGTTCTCCGTGACAGGTACT
Mut= 87 bp
Wt= 94 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
TATAAGATCACCTGGACATCGTGGGCGTTCGCGTCTGGCATTCCACCCTACCTCTGGGTTGGAAAAGACAACCTAGAATGACCTCCGATGAACAGCAGGCATTAGCTAGGCACCGCGAAATCCTGCTTCAAGCAGAAGGAACTAGGCAGGACTAGAACAGACCGGAAGGATCTGCAGTGATTGGTGAGTAAACTGGGAGTCcggtgggaagttagggaaccagcagcgcaggtggagagccagtacctgtcacggagaacgtccgacgaaactacaaccaccacagtgctccgcggcatgacgtctaccagggcccggcctcaggcagccagccactgtgcgcgggggtcacgtggggtcggcgcatcacgtgcgccggtcaggtggcgctgcggggcgggccgacagactgcggggcggacggtggacgcggtttagctcaggtcccaccgatccgacccctgccgtcgccgctgccatgacggggctggagttgctctacctcgggatctttgtggccttctgggcctgcatggtcgttgtgggtgagcggggcccaggcggcccaaggtggcctgctggttggggcggggggcaggtggcagccggagccggccccgcgcggtgccctcccgcgacggaggaggccggccctgggctgcgagagtcacaccggctattccagggtcgcgacgccaggttcctgacctcctctccgcgagccgcatatttgggcgccccgcttcccctttttcctccttgccctcccccacccattcacagcttgttgggtaaacagcgattgggccggcaggatctggcgcggcctggacgcagcgcccgttccctccggcccgtgaaggggcttggtgtcattaagacgggttacctagggagtggccgaaaaaaaaaaaaaaatggaggcctgggtggaggtgctgtttcgctccgggactcttcgcttgtctagatgtaggtggctgacagtttgacagttgggcagttttttttttttcctaggttggggactgtgtctcgaaagagcaaccctttaactcctttcctcattgcttcaggaatctgctacaccatctttgacctgggctttcgctttgatgtggcatggtaagggagggcatggagaggattctcctaggagcctcttctccctgctttggtctgagccccagcccagctcttcagacacacggacctttgggaaatagaaaatctagagatgcaacgtctgggactggtgggctggagctgggtggacactgagttttgtgtggcctccccactcctgccctttcctgtccatagctcaatagctttcttccctgtttccacaccaggttcctgacggaaacttcccccttcatgtggtcgaatctgggcattggcctagcaatttctctgtctgtggttggagcagcctggtgagtgttggagtggcgggacgagtgggaggtgccaagtcgtggcaggtggaatcatcactgggttcttaatcatcctggggacagcaactccttgggctcctttaatcagtttcttacccttcaccaggggaatctatataaccggctcatccattattgggggtggggtgaaagcccccagaatcaaaaccaagaacttggttaggtaagtgtaaaggcttctgcatgtcaaaactagctgcgatgggggtcgggtgacatgctggcttgtgacccgtctcatcgtagcgtgtatgtgagtcagggaaagttggctggggattttctgcagaaatggctgatttccttcttaaagagtttccctccccctcccccaccatttaccatcactgttgccagtattatcttctgtgaagcggtggccatctatggcatcatcatggcaattgtcatcagcaacatggctgaggtaagggaaaaccagcggggacgggtatccattgaaatgcctatgctttccctaaagatcggagggaagaaaaggtaggtgcattaaggaatgccttctgaggacttattctgcccctcacagcctttcagtgctacggagcccaaggccattggccatcgaaactaccatgcaggtaagtgggtaacatagagctcactcggcatggtgctctgggcctttaaccccagtcctgggacgggggtagcaccaagtgagttaagaggccagccagcactacataatgacatagtgagacagacaccctaccacacatacacgcctgggccttacacacttgcttctaccacacgtacacacctgggccttacacacttactcctgcttcctggctccatagctgagagtctcagctctgcagagttggggtgggactggccctgctagcccacactggaccattcctctcctgcttgcctccacctcacgagctacttcttatcctccaggttactccatgtttggggctggcctcacagtcggtctgtccaacctgttctgtggagtctgcgtgggcatcgtgggcagtggggccgccctggcggatgcacagaaccccagcctctttgtaaaaattctcatcgtggagatctttggcagtgccattggcctctttggggtcattgttgcaatccttcaggtgcgagtccccctagagaggaacagaagtgcctcccttttttccccttcccatctcttggtccccctttgtctcctgctctagatgcacgcatctttcctaagtctctccattagcatctctttcctcggctgcctctgaaaatgtctttgtggagtttctgtctctcagggtctgtgttctgtatgtatgacacacttgtcagtagactagcatgtctaaattcatattttccagtgtgtgagtttgtgacttgggtgcttgactgggctgactcttggcgacactaacactgtccatgggacttgtcttctctgttgtacagctgagctcctttgttactgtccatgtctgtttgtctgtcatagtgtgtctgtccgtctgtccacccactggcctgctggttctggtcttgtcactggtcactgtccctgcctgatttgtcctgtttttcctttgcagacctccagagtgaagatgggtgactagatgacctgtatgggtggggccgtgccctactgttatttattgctggttttcctgggacagctggagctgtgtccctcagcctttctggggcttggtgttcagggtctcccctgcactcacctcttgctgcctgttgactctggaggcattgcagtccagactgaaccccagtgtggggagtgggctgctgatggtgactgtgtacagctgtgcacctgtgcccccctcaacccccatccagcccatcttcctagtgtttgtgaaataaacttggtatttgtcccagtcagtgaggcttctgttggccccttgcgacacctttcacaaaggaaggggaggacctgttcactcactccaacctcctgtaactttctgtctacacatcatctctgctctggaccttgctggctcctgggtggcagatcagggtgctaggctggctgtaatgctggcacctcttcattgaagagtctgggctctgagatcctcctagccctatgtctttggacccctgtgcactagacccAAACTCACTACAGCTAATTCTCCTCTCTGTCGCTATACTCCTGAAACTCCCCGCACCTGGATTTTCCTGGAGAACAGTGTTTTACATTTGATAGTCCAGTATGAACACCTAACCCTTCCAGAAACCTCTTCTATGGTCTTAGTTCCTAGGGCCTTGTTCAGCTCCGCCTATTCCTATACAATTCTTGATTCCAACACCA
This mutation is a 3425 bp deletion beginning at Chromosome 4 position 117,884,089 bp and ending after 117,887,513 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 52624 | GGA TCT GCA GTG ATT GGT GA | Common | A | |||
| 52625 | CGT TCT CCG TGA CAG GTA CT | Wild type Reverse | A | |||
| 52626 | CGG GGA GTT TCA GGA GTA TAG | Mutant Reverse | A | |||
| 52627 | Fluorophore-1 | AGC AGC GCA GGT GGA G | Quencher-1 | WT Probe | ||
| 52628 | Fluorophore-2 | CTG GGA GTC AAA CTC ACT ACA GC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 52624 | 0.40 uM |
| 52625 | 0.40 uM |
| 52626 | 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.