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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
>chr5:38225815+38225920 106bp GGCCAGGTGTGGAAAGTCT TGCACTTCACATGGCTTTTG
Mutant= 106 bp
Wild Type = 106 bp
Sequence
Wt Sequence (deletions in lower case):
CCAGGTGTGGAAAGTCTTTGAGAGCCACTAGcatgctgtcagcctagtgaccagccctgggtccctctgcaggggtgatgactacaaaagccatgtgaagtgcatcagtgaaggtcagaagtacggaggcaaaggctatgaagccaagacacacaaaggtgatgcaaaacagcaggcatggattcaggtaagggtccctcccatcctactgctcatccctacctgcttcctcctgggggctcagcagatgggagtccctgagacagcacgtcttagagtgcagcagaagtgtcagaacatcaggaaagataaaattgtgctgcatttatatctggttcgtgtttgctctcaagatatctgagggttcctgctaacttccttttggctgtaaacaaaatcaaagtgtacaggtacacttggcagtggtagaggtcctatagcttagccgtgtcagcctgtcacagctgaacaaagtaaacatttttacaaatttaaggaattttccagtgaaaatatgtacaaaagcagcaagccctggtttgcagcaagagaagatgggtgggcagatgtcacctttgtacagactgcattctcctggaaatcagattagttgttcccccactgaatggctcagagtgagtgtgaacttggggtggtggctctctctgaaggtgtatactgctggcctgaccctcatgagggacttgaaggaggagctgcctggttgagtaccacttgggaagctgctgaaacctttaggatgtgtgtagtgctggcgagatggttgagctcggaccctttcccactgagctgtcctgctgacctttacttaatactgtagtctgccatgtgtaagaacccattatcaggtcagaaggcgcctcctctgccaagggtttttgtgttgttttggtttggttttgtttgtttgtttattggtttttcgagacagggtttctctgtgtagccctggctgtcctagaactcactttgtagaccaggctggccttgaactcagaaatccacctgcctctgcctcctgaatactgggattaaaggcgtgtgccaccacgcccggcttgcaaaggtttgtatagtttcactttttaaaatgtagatccttgattcattttgaggtgttttttgtataacataaggtaagcatctaatttccctgtggggatccactatttctgtcatcatttgttgaaaaggggatctgttctcccatagactcttcttgacatccttgttgaaaagccccttgaccatatcctcaggggtttattcctgcattttctgttcttttccagtggtctttgtggcagtaccacactctttagtttattgtagctttgtagtaagttttgaaatttaaaaaaaaaaacatttttcaaaattgttttgttgtttggagtccatcagggttccatgaattagagcacacatatgctagtctttaccagggctttcaggttaacatctaaatgttgtcagtgcatgaatgccgtttttattcttgtaactaactgtccagtgcccagttccttcaccgagttggttatggttctttatgttgtgctgcagatggaattctcctaatgttcacgtctgaattggtttttttgttgtttggttggttggttggtttttgtttttgttttttggtgtataaaaaagcagcttgttctttaagaggtgagtgtgtgtcctgcagctctgctgaatgcgttgttaattctaactggctgtgtcctgtagcactttctacatgcaagaccacattccctcaaatactgcctgtacacgtgtgacctgattttgttctttctgttaggggggctttggcttctttttcttgcttaaccactttggccaggactccagtgctgtgctgaataggagaatgtttgtcactatgcatagatgtaaatatagagtcttagtttaaaatgtgagagccctttggccatgccaaggacttgagtttggcttcatgtaattatgtcacgtgtttattttcttctatcttaaatagaaaattaatgagttaataaagaaacccaacgtcagccccaaggtgcgagaacttttgcagcaaatcagtgcttttgataatgttcccagaaaaaaggcgaagtttcaggtatggatttaaatgactttcctaaaccatggtgtgacccatctgtggcctgcagaggaacagtggctctttgtcttgtgtccacagaactggatgaaaaacagcctgaaagtgcacagcgactccgttctagagcaggtgtgggatatcttctccgaagcatccagcagtgtgagtcaactccactttgccactgcaatatggcagagaactgggaataaggaaatggggtgccatgtgtggggatcaagatggcatttccagggaAGCTCTGTGCTCTCTCCCTGATGGGCAGGAATGATCTGGGAGCGCCTGTGGTGCAGAGACAGC
A 2382 bp deletion beginning at Chromosome 5 position 38,225,848 bp and ending after 38,228,229 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 47942 | TGC ACT TCA CAT GGC TTT TG | Wild type Reverse | A | |||
| 49612 | GGC CAG GTG TGG AAA GTC T | Common | A | |||
| 49613 | AGG GTG AAT GGC TGT CTC TG | Mutant Reverse | A | |||
| 49614 | Fluorophore-1 | TGC TGT CAG CCT AGT GAC CA | Quencher-1 | WT Probe | ||
| 49615 | Fluorophore-2 | AGA GCC ACT AGA GCT CTG TGC TC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 47942 | 0.40 uM |
| 49612 | 0.40 uM |
| 49613 | 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.