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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
>chr19:42031763+42031872 110bp GTCCCAGTGCCCTAAATACA CACAGGTTCAGCATCTTGGA
Mutant= 100 bp
Wild Type = 110 bp
Sequence
Wt sequence with deletion in lower case
CTCTTTCCTGCTATTCAGAAAGTGTCCCAGTGCCCTAAATACATAGGGACTGGTAGAGTGAAGTCCCTGATAggccagggccagagagggggtcagttacagcttaagtggcttccaagatgctgaacctgtgcacctgtcttacctaggtcgcaatgagcccttgaagaaggagcggctcaagtggaagagtgactatcccatgactgatgggcagctgcggagcaaacgagatgagttctgggacacagcacctgcctttgagggccgcaaggagatctgggatgccctcaaggctgctgcctatgctgctgaagctaatgaccatgagctggctcaggccatcctggatggagccagcatcaccctgcctcatggtaagcaggcagggcctgggctctgtccttacctggactggggctggaggtttcattagcgcagaggtggaaagcaagtttctcatgtgagaagtttctgttttagctggtggagatgaaggagtatgcctatatgtgtgactgtcttcttcgtttaaatgactctttatggcatgaacaaagaagcacaaggcacagaaccaagcgtagtggcacatgcctgtggtcatgagaggtagacacagggggagctgaagttcaagttcaagaccagcctagattacatgagactgtcagaaggaggaaaggaaggagggagggatggacagaatccccctttgcctagtgagaagacaggaagacaccaataaaacctacaagtttaaaaggcactcagggcctgagagcatttgaagcactgatctggaaccttctgctgcattgccgcatttctgttcctagcaccctcattagctgtgaagggaaagtgacagtgaccttcagtcacagacctgggtctttttttgtttgtttttgttttgtttttcaagatagggcttctctgtatagccctggctgtcctggaactcactctgtagaccaggctgccctcgaactcagaaatccacctgcctctgcctccggagtgctgggatcaaaggcgtgcgccaccatgcctggcttcagacctgggtcttgactgtgttcctttctcagcttgtgacttttcctagccatatgtcccagagcagggcaagtctcaggttagagcctgagtctcttgcaatcacaaaggattaatcgtgatgtgcatagtaccacctagtctagtaccagcacttacaaggatactgaagactaaataaatgtgtttccacctgcattatatcataccagtaaggagacagggacccaacccaacacttgcatgtgcattcagccaccacaatgttgaataaacacataagaggtagcatcctgtggctgaggagccagaacctgatgaccaggaggttagccccagtctactgactatggggaagggctagggacagtgaggtagctgtggtgagtgaattaacctgcttacactgttgtagcagaggaccacaggcaggctccttaaacagtagaagtttgtttctcacaatttgagagactggaaagtccaaggtcaaaggcccagctgtttagtcctgatgatgcttcttggtttttttgctgtttttacgtggtaggcgtgcagaagagaggactgggtgggttctctgtttttatttggtaggcatgcaggaagagaggactgctcgggttctctggggcttttccttgaaaggaagctggtcctgtcatttacccgtgtaatcttattccttccaaatatgccttaacatgggagtttgggggacacagcattcagtgtgtgacatcaggccccccagactacactgatcaccctgcctttcccacaggaactctttgtgaatgctatgacgagctgggtaaccgctatcaactgcccatctattgcctgtccccacctgtgaacttgctgctggagcacacggaggaggagagcctggagcctcccgagcccacccctagtgtgcgccgtgaattcccactgaaagtgcgcctctccacaggcaaggacgtgaggctcaatgccagcctgcctgacacggtggggcaactcaagaggcagctgcacagccaggagggcattgagccatcctggcagagatggttcttctctgggaagctgcttacagaccgcacaaggctccaggaaacaaagattcaaaaagacttcgtcatccaggtcatcatcaaccagcccccaccaccccaggactgacggactgacggactgacggcaccaactcctaggaaaagaggccatggggggccctgcaggctgtggggcccagtcctgccgccttccagtgctgctgtcttcttcaggggcgcctcccaccgccctgctgtatggctcttagtgagctgcgaagggacttgccctctggggcactggatccaccagtgctcagcacccagggagcagtgctgcctgccacacacaggccttgccaaccttggagaaaaggcacatggcactctccaccacaggttggagccatgggggccgtcctggcgacctgtcatctggtgccacgctgtgctcactccagttttctgctcagggttggaagcaattgctgtctcccctttatccctcaccatgactctgccctgggcacagtgccagtcccctggatggggagggagcccggtgggccccagggcttaaggagcttcaggtgggcctctctgcctctccctgcacccagcacaatctgcagagatgaggcgggtggctggatagccaggcttcatggcaggggtttgcacagggctgtccccaactgtaacccaggcagggtggggtccgaagcctggtcatagcccagcggggccctgtgtacagacacatctgcattttatcaataaagcctttgctcctctcttgcctagcacatgacttcctgtttcacagcacgccagccagccggagcccagagcttctgctggcttggctgcagatgggcacagcagagcatccaactgatgctcctcctgGGGGCCCTAGTCTCATCCAACCTCGCTGTGTTCTGTGGGTCCTGAAAAAGGCCACTTCACCTCTGAACACAGTTTGCT
A 2945 bp deletion beginning at Chromosome 19 position 42,031,812 bp and ending after 42,034,756 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 44266 | GTC CCA GTG CCC TAA ATA CA | Common | A | |||
| 44267 | CAC AGG TTC AGC ATC TTG GA | Wild type Reverse | A | |||
| 44268 | CCT TTT TCA GGA CCC ACA GA | Mutant Reverse | A | |||
| 44269 | Fluorophore-1 | ATA GGC CAG GGC CAG AGA G | Quencher-1 | WT Probe | ||
| 44270 | Fluorophore-2 | CTG ATA GGG GCC CTA GTC TCA T | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 44266 | 0.40 uM |
| 44267 | 0.40 uM |
| 44268 | 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.