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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
>chr14:66303913+66304015 103bp GCATTGAGTTGCTTTGCTGA GTCGTTTGCACCTACACCAG
Mut = 118 bp
Wt = 103 bp
Fam = Mut
Hex = Wt
Sequence
Wt Sequence (deletions in lower case):
GGGTGGGTAGGGGGCAGTATTTAGCATTATAATACATAGCAAAAGACCAAACCTCAACATGGCTGGGCCAGGTAGGAAGCCTGGGAGGACCGAGAGGGGAGGGAGGATGAGTCCCTGGCATTGGCCCACCCATCTCTGTCTTTGGGGCTCTTCATGTCACACCTAGAGCATTGAGTTGCTTTGCTGAAAGTGGTCCCCAGctggggcgtcccaaagccattacatctgaccttgcttgcttctccctcctctggtgtaggtgcaaacgacttggctggaaggccgcatccgggatgagtttgacaagctccgtgatttcctgagggtggaggaacaggccaccttggatgccatgaaggaagagagcagaaagaagcacctgcaggctgaggagaagatgaagcagctagcagaacagaccgaggcgctggctcgggagattgagcgtctgcagatggagatgaaggaagatgacatgaccttcctcatggtaagaggctcatcgtgtgaactgagggtggggtcagggtcaggggccagcagctcagaccacagaccatgttcaacagtctttctgtgatggtggagggccttattggcatgccttgtggcagagcaaggcttggcggttgagaataccctcttaccttcacctccgccccttactgttgctgctttcttattctttgattggttatctctctatctctaatgaaatagtcaaattgtgtaataatttaagctttaaaatttttattacatttttatttatttactgtagatgagtgtcttagggttcatttactatgaagagacaccattaccacagcaactcttataaaggaaaatgcctaactggggctggcgtacagttcagaggtttaatccattgtcattatggtgggaagcatggcagcacacaagcagacatggtgctagagagggagttgagagttctacatctggatcagtaggcagcaggaagagagagtgagccactgggtctggcttgagcttcatccccctgtgacacacttcctccgagaaggccacacctcctatactgcccctctctatggacctatgggggccatgtcaaactgtctctatgggtacacattgcagagtgcctgtatggaagtcagtcggtcttcttttaccacgtgggtcccacagaccaaactgaagttgctaagcttgacagcaagcaccttaacctgctgagccatttagctggctccgttgttgttgttgtttgttttgtttatttgttgctttgttgtttgctttctcctctgtctttcttgcttttctccctccctccctccctccctccctcactgtgcagtgctggatctgtagcttaggctttgtgaatgacaagtactctcactgaggtatcttttatcttttccttagttttggggatgggtgtgagggtacaggattgcactctgccttctttctctaacgctttcaggctgaggagctggctcagcctgtgaagtaggaggagccagtctgatccctagatgccacagaaaataagctgagcatggtggctcactctgtttgtttttaagctgaaattattatacaatttgactattgcattcaggagttgagaacaaccaactaaccagccaacaaaaaaagccactaagtggcctgccagcctagcctgcttggtgcgatccagggcagtgaaacacactgtactaaagaacaaggcaggaataagctgtggtttacctctggccccatgagacgtatgtattaatgctgcgcgcgcgcgcacacacacacacacacacatgagtgcacaggaaagaaattctgttcttcctttaaaaacatgattcattgtctttcttccctttactgattttgtccttccttgcagagcagtttcccagacttgacagctagtttatcagtcagccccctttgatggcgagtgttcccctcgtgcatggcgtttttacctgtcagttggccttccgttctcacttgagttacttctgcttttgctgtttatgggcagtaagactctaagcgtgtgtgtactggatttgtgtggacatgaacttttcgatgtgtacccaagggatttctggattacttgataattctgctttaacccattaagggctgctaggcagttttaccaaagtagctacactattttgtcttcccaccagcagagtctgggtgtcaagttcctgtacctcttggccacttcttttctccatagctttccttgtgagtcacttgggctctcattgtgcttccagtgtgcactctctggatagctgatgacactgggcatctgtcttttcagtggctttgtggacattactagttcttgcttaaatgtctgtttaatttgtttatacagttttttccttcctttttttttttgttttggttttttttgtttgtttgtttgggttccccccacccccaagatggggtatctctgtgtaggtagccctggctgtcctggaatttactctgtaccaggctggcctcaaactcagaacacctgcctctgcctcccgagtgcaggaactagagatgtgtgctaacgtgcccagctatccagttttcagtagggttgtctgttcattgtttagctgtgagagttcgttccttttctgctccagtatgactcttacctcttccgtcttagatttgcatttaaatcttacatgtattttaagggtgttttgttttgtttttgtttttgttttgaatctgacacaaggcagaggctttccccattttggttgtattgacatgttctctgagaaggcagttctattaacttaccttggtcctgtagaaactccagcgttatctagactgtcacttctgtcctcattactcctgatatctctaccacagcagcttgtgtaacaagtgtgaagtttgggagtatgagtcctccaacttggtggttttcaaggttgcttttattcagcatttcctgaaactccatatgaactttaggatcagttttaagttttgccaaagaaggcccaaggccttgtgtataccgagcaaggtctccctcccatggctgcatcctcaccatgggactttcaatgggaggtgagggggggacactgaatctgagatgagatccccatggggtatgtgacagctggatgatgtcagttcttctcatccatgagtgtgggacatctggcaattcttggtcagaaattacctgcagtttcttagtctttgtctctgctttttttttttttctttccagaaacacaagagccgaaaacgccggtaagttggcaaacccaatggggatgggaaagggtaaatagatagaaagccaggccttcactgtccagtggtggaagggctaggcaggtagtgtgtccaactgggtggaatccagagggagctgtagaggccagccaaggggagcgctttaaagacatcccagttctggttggcatggagacagggagttgtttcagaacatagagggtcgatactctccacagGGATTGACAGCACCAGGCACATGACTCATGTTTCTCAGTCGTCAACAGGCTTGGTTTTTGGCTGGCTTCCTACCTCTGGGAAAGAGCGTTGGCATGCTTTGCTGTGGGTGGATGCTTGCTGGGCTCTCCAAGATGGGTA
This mutation is a 3327 bp deletion beginning at Chromosome 14 position 66,303,946 bp and ending after 66,307,272 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 51870 | GCA TTG AGT TGC TTT GCT GA | Common | A | |||
| 51871 | GTC GTT TGC ACC TAC ACC AG | Wild type Reverse | A | |||
| 51872 | TCT TTC CCA GAG GTA GGA AGC | Mutant Reverse | A | |||
| 51873 | Fluorophore-1 | CGT CCC AAA GCC ATT ACA TCT | Quencher-1 | WT Probe | ||
| 51876 | Fluorophore-2 | CAG GGA TTG ACA GCA CCA GG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 51870 | 0.40 uM |
| 51871 | 0.40 uM |
| 51872 | 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.