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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= 114 bp
Wild Type = 109 bp
chr7:4921736+4921844 109bp TCCAGTGCTTGCTCATTCTG CCTGGCTTTTCCCCAGTTC
Sequence
Wt Sequence (deletions in lower case):
TAAGGAAGAGCCCCCACAGCATGGGCGACACAGGGAGGCTGCCCCTGTTCCTTGAAGAGCCGCTGTGAGTGGTGAGGAGGTCCAGTGCTTGCTCATTCTGATCAGAATCCTTTCACCAGGTGTGATGGCCGGCTCCCacgtggacatggcgccagcttccaccaccgagggaactggggaaaagccagggcccactgcgccagcacccaccccagctgctcagtatgagtgtggggaatgcggcaagtccttcaggtggtcatctcggctcctgcaccatcagcgcactcacacaggtgaacggccctataaatgcccggactgccccaaagccttcaagggttcctcagccctcctctaccaccagcggggccatacaggcgagcggccctatcagtgcccagactgccccaaagccttcaagcgctcttccttgctgcagatccaccgcagtgtgcacacaggcctgcgggccttcacctgcggccagtgtggtctggccttcaagtggtcatcccattaccaataccacttgagacagcataccggtgagcggccctacccgtgccctgactgccccaaggccttcaagaactcctccagcttgcggcgccaccggcatgtgcacacaggagaacgcccttacacctgtggcatttgtgggaagagctttactcagagcaccaatctgcggcagcaccagcgggtgcacacgggcgagcgccccttccgttgcccactctgccccaagaccttcacacactcctccaacctgctgctgcaccaccgcacccacggccctgcccccggccccgcccctgcccccgcccccccaggggagaccagcagagctgacaccaaggttctggtctctgatgcctacttgcagccccgtagcccgcctgagcctccagccccaccaccccagcccccacccgtggtgcccgagctttttttggctgctgctgagactacagtggagctagtgtaccgctgcgatggctgcgagcagggcttcagcagcgaggagctcctcctggagcaccagccatgcccggggccccctgtagccacccagtcccaggatgtgcctgccgagctgccccaggctgactccgccctaccacagcccccacctgccactccaggccctcctaactttgcttgtcttccttgtgggaagtccttccggacggtggctggcctgtcccgtcaccagcacagccatggtgcggccagtggacaggcctttcgctgtggcagctgtgatggtgccttcccacaactggccagcctcttggctcaccagcagtgtcacgtggaggaggcggcagccggacgcccacctccccaggctgaggttgcggaggtcacctgtccccaggaacctgtagccccagccactcctgccccaccccctcctcctccacctgccccggtggtttctgcagagcggccctacaaatgtgccgagtgtgggaaggccttcaagggttcttccgggctgcgctaccaccttcgggaccacacaggtgagcggccctaccagtgtggggagtgcggcaaagccttcaaacgctcctccctgctggccatccaccagcgtgtgcacacaggcctgcgagccttcacctgcggccagtgtggcctcaccttcaagtggtcgtcccactatcagtaccatctgcgacttcactcgggtgagcggccctatgcctgcactgagtgtggcaaagccttccgtaacacttcgtgcctgcggcgccaccggcatgtgcacaccggtgagcggccccactcatgtagtgtgtgtggcaagagcttcgcacagacgtccaacttgcggcagcaccagcgggtgcacacgggcgagcgccccttccgttgcccgctctgccccaagaccttcacacactcctccaacctgctgctgcaccagcgcactcattctgccgagcgcccttttgcctgccccatttgtggccgtggctttgttatggctgcctacctgcagcggcatctgaggacgcacaccccagccaccacaacttcgggcaccaccggctctgctgtagcatcgcagccccctgccccattggctgcagctccaactccactggctgctcaagatgttcacgtactccctaacctacaggccacactttcacttgaagtagctgggggaacagcccagcctacgcccccaggcccggctgctcccagttctcaaacatttctcctggtacagactgcccagggcctccagctgattccaagcagtgtccaatcccctactcctccacccccgcctccaccccctaaagtcattctgctgcctcctgccagtgctggaggtccgggcagtggtgctgcaaggccaggccctcggtctgttgggaaagctggacaggggacaggggtggtgtggtttccaggcccaggtggtctggggctgcaaggaggggccaatgcaggggctagcgggggagggcagagcctcattgttctgcagaatgtagggagtggggagacaggaccacaggaagtgagtggggtccagcttcagccagcacaggaagtggccacggtacaactacagcctgcacaggaagtgactacagtccagctacaaccagcacaggaggtgactacggtccagctccagcccctgacaggccaagtctccaattctaatggaggagctggcactacagaagctccaaacctgctgctggttcagagtggggcaactgaagagttgctgaccggccctgggcccggggaagtgggagacagtgaggccggtgctggtgtggtccaggacgtcctgtttgagacactgcagacagatgaggggctgcagagtgtgctggtgctgagtggggcggatggtgagcagactaggctctgcgtgcaggaggtggaaacgctttcccccggtctggcggagccagcggccactggcccatcaggacagaaactccttatcattcgtagcgccccagctactgatctgctagaaaatagcagcgttgcaggaggcaccacaacactgcaactcctggccccgtcagcacctggcccggtctctgcccctgtgggggtgcccgtagctcccccgtcccagatggtacaagtggtccccgcagtggctggaccaggggtcatggctccccagaacttgccctccatccagattgtgcagactcttcctgcagtccaactGGTACACACGTTTTGAAGAGACCCCCTCCCCGCAATTAACTGTTCTCCTCAGCTGGGCTGGGGTCAGCCCCAGAGTCAGGGGGGCTGCTGGCTGGACTTGCTAATAAAGACCCAGAATCTCTTCCTGGTTTTGTTTTCATGGGGGAGGTATAGTCATGGGCATAGCCCCTGCTTCAGACCTGGAAGTCTTCATTCTTGCTTCCCGGATCTGAGGTGACTTGCTTTGTACTTGGAGGGCTAAATGCTT
This is a 3088 bp deletion beginning at Chromosome 7 position 4,921,793 bp and ending after 4,924,880 bp (GRCm39/mm39).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 60195 | TCC AGT GCT TGC TCA TTC TG | Common | A | |||
| 60196 | CCT GGC TTT TCC CCA GTT C | Wild type Reverse | A | |||
| 60197 | Fluorophore-1 | TGG ACA TGG CGC CAG C | Quencher-1 | WT Probe | ||
| 60198 | CCC AGC TGA GGA GAA CAG TTA | Mutant Reverse | A | |||
| 60199 | Fluorophore-2 | CCG GCT CCC GGT ACA CAC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 60195 | 0.40 uM |
| 60196 | 0.40 uM |
| 60198 | 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.