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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
>chr1:37882850-37882964 115bp GGCATGTGATAGCTTTTGAGTC CATGGGACCAAGTGCCTCT
Mut= 98 bp
Wt= 115 bp
Fam=Mut
Hex=Wt
The WT Reverse primer (primer 64370) anneals over the nucleotide sequence containing mouse genomic variations rs585271614.
Sequence
Wt Sequence (deletions in lower case):
AACAAGAAAAAATTTCAATTTCAAGAAAAAAATTTAAATTTAAAAAAAGGATAGAGCCCTTTCTTAGCTTCTGCGAAGGCAGGAATCGCCTTTGGAATAGTATTTCATCTTCTTTCCCTATGGGTATGAAACATTTTGCAATGTGCAGCCTAGAGTAGGCATGTGATAGCTTTTGAGTCAGTAAGTTTAAAAGATTGTAACTTATCCTGATTgtcccctgtgtgccatataactgtgtataaagaaagtgctcagaggcacttggtcccatgggtagaaccacctgccaacaaaggtaaagaccagagttcaatccctgggaacccacatacaccccccccccccccacacacacacaaaagattagagatgtaactttttaaagtggtccctggttacgttactgtagcttgtgttttgcagatggaaaatatcacaagcaaattaaaatagaagagaatgcaactggcttcagttacgagtcactctttagagaatacctacacgagacagttacagaagtttggatagaagacccttatattagacaaactcatcaggtaagtttactaagatagaatgaaatgtgactttaaatgtacagtcagctacctgtgtttatgggtttcctacccacggattcagtggaggatcagggatataggtaaaaattccatctgtactgaacatatacaaacataacaagacaagggtaacgaatggtgtttacattgtgttaggtatcatgtagtctagaggcgatttaaagggcccaggaggaggtatgtgggttttgggatgatactatattgcttaaaataagggatggaaccatgtgtggatatcttagtatctggctaagggagggtatcctagaatcagttctcttaaatagttttgttagaccttggagtttccttcaggatacagtgcttataaagtacagtgttttattttgctccctgtgcacagtgcccagtaatttgctgtagaagcttaaatttaaaacttccatgtagcttgctatatttgggtccatcatgtgtttccctgtggaacagcgcaccatctttactcaaattattttaatccacctctttggtggtgagccctgaagaagcaccaccagatgggggagctcctccaccagggaggcccccaggcattcctccaggcgtgccaacagctctgctacagcttggtaacgatgggactgcaggcgttcagattcttccttctccgcagcctggttcggatccggctggctgaggattatagactcagttcttgccggagcactgtgaagctaggactaatccaccctaatgcctttcagctctataacttccttcggttttgtgagatgctgattaagaagccgtgtaaagtaagaaccattcaccttctcacctctgggtatgaagtaggtatctgaaatgcctcattctttcttaacttactgtgtccgtcatatgtgacctgatcctacactgtgtgccaagcatagactgtcttaagaaaaaaagctgttgatttgagagtgttgacctcgttcgcattcagccattttgctgagctctttgaatttgagtttgtcagggtggtctcagtggccctgcagtctgcaccagcgactcttctgttctttactccatgtgctgggtttcgactgtggctgtgacttccttgatttagacttgagtagaagtgtatctgaagaattatactttccagggttagtctaagcagtaggttgtctcacacggataactgagcatccttgctaggatacgtttaagaagctgagcatggtgccatgtgcctgtgctcttagcaacagggaggctgacgtaggagggagttgtgtgatagtctcaattgtttaattgtttatataaaaacaatataaaaagaaaagaattacccaaccaacagttgcttctttgagttagcaatattgaaattgatataaatcttttcagtttaatctctctgtagatttatttatttattgtatagctcatggatagttaataacagaggcagtaaattatgttttcatgctatcaatagggtttggggaacacacagcaaagtagtggcctggaagaaataaagcagtccctcgggagtcacggagtggtgctggaaataaactactcttcatctatacatgaccgagaaattaggttagtgtataatgttttactttttttgtagctgagatatactttaaaaattctaatgtaatataattaaatacatgtatttaaatattcaaatcaagctcaaatttagtaattgtatattggacaaaaacaaacaaacaaaaaaaacccatcttggtttacctcttagagtgtgttaattcttagtcaagtaatatttacaggtcctatgtcatGTGGGAAGGAGTTCTGAGTTCTGTGCAGACCATAGCCACTGTTATGTTAGGAGATAATTCCTTTCCTACTGAAATTAGTTTACCTGGTGTGGCTTTGGTGGTATGTTCTAGTAGTGTACTGTCGATGAGAAAATGCATGCACAGTTTCTTAGTGATCCTTTAGCATAGTAGATTTCATATGCTACTGCAAAGAGACCCTGTTTCTAATAAAAAGTAAATCAGAAGCTAAATATGTAAAGTGG
This mutation is a 2192 bp deletion beginning at Chromosome 1 position 37,880,718 bp and ending after 37,882,909 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 64369 | GGC ATG TGA TAG CTT TTG AGT C | Common | A | |||
| 64370 | CAT GGG ACC AAG TGC CTC T | Wild type Reverse | A | |||
| 64371 | AAC AGT GGC TAT GGT CTG CAC | Mutant Reverse | A | |||
| 64372 | Fluorophore-1 | CCC TGT GTG CCA TAT AAC TGT GTA | Quencher-1 | WT Probe | ||
| 64373 | Fluorophore-2 | CTT ATC CTG ATT GTG GGA AGG AGT TC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 64369 | 0.40 uM |
| 64370 | 0.40 uM |
| 64371 | 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.