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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
>chr1:171277214-171277302 89bp CCACTGGCAAAAACTAGGTAAG TGAAAGCGTTTTAGCTGAGG
Mut= 93 bp
Wt= 89 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
CGGAGGTATCAGTGGATTGGCCGCAAGTTATCATCTGATCCGAGGCCCCAGTCCTCCTAAGGTGAGTCCCCACTAGTACCAGAGAAAGACTCATTTAATACTCTTGATATTTCTAGCAAAAGCTGCATAGATTTTTGTTCCGTGAAATATGCACTTCCCCCGACCCCCAGTCCCTTTGCCCTGTGgcgacagtgcttcacttttggcgccaggtgatcttagtggagggcagcaagcgtttgggaggatggatccgctcaatccgaggatcagatggtgcgatctttgaacttggacctcgagggattaggccggctggagccctgggagcccggaccctgctcctggtgagcgtcttgtggaatgtgtaggacaggtagtggaggagagttctccaaagggacggagttgcagagaggaaaaacgttgggtcagtttttccttagtttctcctcttcctgagggcttgtggagaacaggtttctgaacttggcttggagtctgaagtcttgcctgtccgaggggatcacccagctgcccagaacaggttcctgtatgtaggcggtaccctgcaccctctaccctccggcctcaggtaacacctcactattacacacacccccctttcgctccctgtcaggccttccagccacagggggtctaatggctgaaaagaaccctctgaaaggtgaatctgggagttttcttttcaaccatgccttcctccatgcagggggctactccgtccttcaccccccttctcaaaacctctgttttgggctgggctgagggagttgctgaagcccaggggcaaagagcctgatgaaactgtgcacagttttgcccagcgccgccttggacctgaggtgacattgctcagaggtggtctcaaacctcttcccctcctgttgctgagtctctaactcagacttaaccccggggactcttgaatcatttaggaatgtccactatctattgctggaatctccaaaacccagtgttgggaataaatgccttcctttccttcccatccttttgttacagtgggaacatctccaacccaaactctctccttcccttagtctctcacaagcagggccaatggaaatcagtcagggtggcttattccttggtccctcagtcccagtcttactcttaaggtggcatctttagccatggacagtctttgccgaggagtatttgctggcaacagccgggagctcagcatccggtcctgctttcccagtctcttccaagctgaacaaacccaccggtccatattgctggggctgctgctgggcgcaggtgaggggcagcattgattggttcagagggtgaaaaaaaaaagcctgaggacaaactgaggtactgtgtacaaggggtactagtttaatcttttacccacccacccctccttttaggacagagtccccagccggattcctcactaattcgtcaggcccgggctgagcgatggagccagtggtcactccgtggagggctggaggtgttgccccaggctcttcacaatcacctagcaagtaaaggggtcactgtcctcagtgggcagccagtctgtgggctcagccttcagccagaagggcgctggaaggtaggagaacctctggagtgtagcgaacctacgtcagtgttactgcttttattgttgttatatcggggattgcaaaatttcacctggcaaaaagtttcatatgcaaatgtacacgacctgattttatagcaaaataggccttcggggggaggaggggggcttctttgctcaacatccatttctttaagtttcgtccaagcttttaaaacatcatgataccatttcctcagacagccaccgttctaatactctgtcgcctgcgagctgccatggctgcacacacatcagtcacaagactcataaagtctttgctaagccctgcggtattcctcagatgggtctgcctcctaggtcaatgatacctttttgtccataaatgtcttcctctgggaagagagaaagctgaaggaagagctcagcagttgcgagcacgtactgcctttgcagagggcctgacctccacaggtgctcacactcaccactcacgcgcacacatccacagactatggcaccatctttgtcagagtggctgaccaggtcaggttaaacccttagcattggacgtactgttttcctgccttagggaagctctgtagatactggctacctcaggttgctaggctctatttgtgaaccttcctcagaatgtccttaggtaaagcctgtgagcggaagtagtgctttcgacgtgacagggacagagccacccaccttcactgaacattccagagcaggcgaactggcccactttctggattacacagagggtttcttggtttttgaagtgatcttgtcactcatatccttcatcctgggtcagcacccagtctttcctacctcttgagtacctctgaaaccgaggcagcaaatctcaccttccccacaaagtcctcataacaccgtctttcaagttttcttctcactcaggtgtctctaggggacagcagtctggaggctgaccacattataagtgccattccagcttcaggtaatgggacagcccttccctttcctagccattaaggggacaatagtgtgccttcatatgccagcagggccacacaggctcctgggctgggctagtaagacagagctagcctagaatgaacgcctgctctccagagctcagcaagctgctccctgccgaggctgcacctctagctcgaatcctgagtaccatcaaggcagtgtctgttgctgtggtgaatctgcagtaccgaggagcttgtctgcctgtccaggtataagaggaagaagagtggagacacgggctcggtgacagccattccctgtgacccacagacatcttcttctccagggatttggacatttggtgccatcgtcagaagacccgactgtcctgggaattgtgtacgactcggtggcttttcctgaacaggatgggaaccccccaagcctcagagtgactgtgagaggaagcatggcttggggtggggtgtccatggggcttctctgtgctgcagaataggcaagggcgggctggtcagtgctgcgtctcctcgtaggtgatgttgggaggttactggctacagaagctaaaagctgcgggccaccaactgtctccagagctgttccagcaacaagcccaggaggcagccgccacacagttaggactgaaagaaccaccgagtcactgcctggtccatctacacaaggtgagtcaaacttactcattctccgctgaaagccctggaagtgacgacaggcatgttaatcaagccaagggctcggaagtcactcataaactttcccttgcatcttctctctccttagaactgtatccctcagtatacaataggccactggcaaaaactaggtaagctgggcgagctgctggacggaCGAGCGGCTGTGCTGAGGGTGTCAGACCTCAGCTAAAACGCTTTCACCCTTACCTTTCAGACTCAGCTATGCAATTCCTGACGGCCCAGAGGTTGCCCCTGACTTTGGCTGGAGCCTCCTATGAGGGGGTTGCTGTCAATGACTGTATAGAGAGTGGGCGCCAGGCAGCAGTTGCTGTCCTG
This mutation is a 3290 bp deletion beginning at Chromosome 1 position 171,277,260 bp and ending after 171,280,549 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 47345 | CCA CTG GCA AAA ACT AGG TAA G | Wild type Forward | A | |||
| 47346 | TGA AAG CGT TTT AGC TGA GG | Common | A | |||
| 47347 | TCC GTG AAA TAT GCA CTT CC | Mutant Forward | A | |||
| 47348 | Fluorophore-1 | CGA GCT GCT GGA CGG A | Quencher-1 | WT Probe | ||
| 47349 | Fluorophore-2 | CCT TTG CCC TGT GCG A | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 47345 | 0.40 uM |
| 47346 | 0.40 uM |
| 47347 | 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.