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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
>chr12:28677478+28677587 110bp GACCCAGCAGGACTCTAGCA TGCTTATTCTTTGCCCATCC
Mutant= 110 bp
Wild Type = 110 bp
Sequence
AGAGGATTGCCACTCCAGTCCATCTCAATGAACTTGAGCTTGGTCTGTGGGCCTGGGAAGCATGCTGACCACAGAGAGTCTAGTGAGACTTGCTGCATGGAAGATGGCAGAGTGGCCACACACACTGAGTGAGTGTTGTCTTGGCATCTGTGAGGAAGAAGGACTTCACCTGCAAATGTGGAGGACCCAGCAGGACTCTAGCAGAACTGGAATTTCcacctggcactgtgaaaatggatgatccatcaagagaggaggtggagggctgtgctaggatgggcaaagaataagcagtgctctgttaggatgatccagccacaggtctggaaggacgtgttgctatgtttccatcacagggccggggagcagacctagagccttgtgcaccactaggcaagcactataccagggagttatactccccgcttagctgctatgcatccttgtgctaaggtgacctccactttgcttaaacttacatagctagatgctgacaagtatgagaacgatccagaactggaaaagatccggaaaatgagaaactactcctggatggacatcatcaccatatgcaaagatacacttcccaattacgaggagaaggtgagcaggccctgctgggagggaatccttaccttcttgcctgctaacacaagacttttccgtgtggtttggggcagagtgatgctttgtaattgtcttagttaatgaggcatactgctgaatgatactcactctttctagttctttctgaactctggctggttcaacccagctgttctggctcaaaaattcttttcaagctgactaattcaaactggcttctcttagctcctgactgaaaaaatctctgcgtggtctcaaactaactctggcaatctgttctaattttctgcaaccaaactatgtaaaactgcctcctccagaatcaccatgcactgctcgcgctcgctctctctctctctctctctctctctctctctctctctctctctctctctctctctctctctctttcccgagacagggtttctctgtatagccctggctgtcctggaactcactttgtagaccaggctggcctggaactcagaaatccgcctgcctctgcctcccaagtgctgggattaaaggcatgtgctgccaccgcctggctatgcactgctctcttaaagttacctctttgctgttctagtgagagttgggcgtaccctatctctgactcattctgtgaaatctctctctgattcatcactttgtctgcctctcagacgccattttcaaacatggttgcttccttctgtaaactaaccctcctttcattgtctgggattaaaggtgtggactaagggcatgtttgtatctcaggcagagggattaaaggtgtatgatgtgtctgcgttccagctggatcacacaagcctaggtcttggatgtgatcccttgccagagcacggatcatgttgctgggttaaaattcctttacaatgctgttctcatagaatgtgctggggtgattccctttctttcagctcttttgaggggtatgaatctggtggtactgtgtcccttatgtgtggtagatccaccagtgatgcttggattggcttggggttttcttcatgggattcacagctctgcatctggctctgcccttgacttgttgtcggttggacgatacatttttctgtgagcatctgtgttgtcttcggggtgggtacctgaccacttcactgggaccatttgagcttagtggcatagttatttagagtggagcatggtctcaggtaggtaatctcagcttctggtaggtgaagacaggaggagcagaagttcaaggtcatctcagctacttagcaagtttgaggacagcctgtgctacatgagaccttgcctcagtttgcccctaccacttgggttctgtggattaaatttaagacatcatctcggtggcaggtgcctttaccgacagtgtcatttcctggcttggctcttgacacttttaataccaagaattattcactgtagtctagctgtaagggctgggtctgtgtgatcggtctccaggaaactcacaggttcccccttgcactagatcaagatgttctttgaggaacatctgcatctggatgaggagatccgctacatcctggagggtagtgggtacttcgatgtcagggacaaggaggacaagtggatccggatttccatggagaagggggacatgattactcttcctgccggcatctatcaccgcttcacactggacgagaaggtgagagtgtaccatggtgtggtctgtgcatagacatcgcaggtgaccatgatgacccactaggtgtgttgtgtgaggctcatcgcctagatacatggccattcacagctctttcagagaaagccagaagagcttgtggaaccagaagagcctgtggccatggaacctgggagggaagggtcagaatgtcgcctttgttcttgttggacagcgaggcagcattatctacatggctttggtcaaggggcaggtatcttcaggagggagagctcctgggggggacctgcacttgtcactgACTGGGGCTTGTGGTGTAGTGTGTGGGTGCTCTCATCTGAGGAGCTTCTCAGCAGTTTTGCAGTACATGCTTCAAGGTCACCCGCAGCTATATATCAAGTTTCAAGAATAGGATGGGCTAGATGAGACCCTGTATCAAAAAAACAAACAAAAACCCCAAACAAGCAAACAAACAGATAGTTACAAATGATGAGATGGGTACAGGGTGTTTACCAAT
A 2357 bp deletion beginning at Chromosome 12 position 28,677,511 bp and ending after 28,679,867 bp (GRCm38/mm10). In addition there is a 20 bp AGATACTTATGCTGCCTAGC insertion at the deletion site.
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 45107 | GAC CCA GCA GGA CTC TAG CA | Common | A | |||
| 45108 | TGC TTA TTC TTT GCC CAT CC | Wild type Reverse | A | |||
| 45109 | CCT TGA AGC ATG TAC TGC AAA ACT | Mutant Reverse | A | |||
| 45110 | Fluorophore-1 | CTG TGA AAA TGG ATG ATC CAT C | Quencher-1 | WT Probe | ||
| 45111 | Fluorophore-2 | TGT AGT GTG TGG GTG CTC TCA T | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 45107 | 0.40 uM |
| 45108 | 0.40 uM |
| 45109 | 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.