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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
>chr10:127192598-127192705 108bp GATGCTGGTCTCCAAGGATG TGATCTCTTACCCCCTGGAC
Mutant= 109 bp
Wild Type = 108 bp
Sequence
Wt Sequence: GAAACAGAGGTAACACAGGGGGTTCAGAAGGAGCCTGAAGTAAGAGGCACATTTATCTGTCAAATGGGGACTCCCCACTCTGAGTCCCTTTCACCCTGGGGGCAGTgtcgttcgtcctgtccagaatggcagcctgtggaggctcctgcaagaacaaagtgactgtctccaagcctgtgtgggacttcctgagcaaggagaccccagcccggctggcccggcttcgggaggagcatcgggtgtccatcctcatagatggcgagacttctgacatctacgtgctccagctgtccccacagggtcctcccccagcccctcccaatgggctctacctggccaggaaggctctcaaggggctactgaaagaggcagagaaagagctaaagaaagcccagaggcagggggagctcatgggctgcctggctttggggggtggaggggaacaccccgagctgcaccgcccaggccctcctcccctccgagcagccccactcctgcccccaggggcaagagggctaccccctcctcctcctcccctgccccctccactccctcctcgtctcagagaggacgctgaagagcaggaaaccacctgccccatttgcctgggggagatacagaatgccaagacgctggagaagtgcaggcactccttctgtgagggctgcatcacacgggccctgcaggtgaaaaaggcctgccccatgtgcggccgattctacgggcagcttgtgggcaaccagccccagaacggacggatgctggtctccaaggatgccaccctcctgctgcccagctacgagaagtatggcaccattgtcatccaatatgtcttcccgcctGGCGTCCAGGGGGTAAGAGATCACAGTTCCTACCCGTTGGCTTCCTCCAAGTTCACTGTCTCTTGACTTAAGAAAAAAAGGCAATGTAGAGAGTATGTTGTTAG
Deleted Sequence: GAAACAGAGGTAACACAGGGGGTTCAGAAGGAGCCTGAAGTAAGAGGCACATTTATCTGTCAAATGGGGACTCCCCACTCTGAGTCCCTTTCACCCTGGGGGCAGTgtcgttcgtcctgtccagaatggcagcctgtggaggctcctgcaagaacaaagtgactgtctccaagcctgtgtgggacttcctgagcaaggagaccccagcccggctggcccggcttcgggaggagcatcgggtgtccatcctcatagatggcgagacttctgacatctacgtgctccagctgtccccacagggtcctcccccagcccctcccaatgggctctacctggccaggaaggctctcaaggggctactgaaagaggcagagaaagagctaaagaaagcccagaggcagggggagctcatgggctgcctggctttggggggtggaggggaacaccccgagctgcaccgcccaggccctcctcccctccgagcagccccactcctgcccccaggggcaagagggctaccccctcctcctcctcccctgccccctccactccctcctcgtctcagagaggacgctgaagagcaggaaaccacctgccccatttgcctgggggagatacagaatgccaagacgctggagaagtgcaggcactccttctgtgagggctgcatcacacgggccctgcaggtgaaaaaggcctgccccatgtgcggccgattctacgggcagcttgtgggcaaccagccccagaacggacggatgctggtctccaaggatgccaccctcctgctgcccagctacgagaagtatggcaccattgtcatccaatatgtcttcccgcctGGCGTCCAGGGGGTAAGAGATCACAGTTCCTACCCGTTGGCTTCCTCCAAGTTCACTGTCTCTTGACTTAAGAAAAAAAGGCAATGTAGAGAGTATGTTGTTAG
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 37338 | GAT GCT GGT CTC CAA GGA TG | Wild type Forward | A | |||
| 37339 | TGA TCT CTT ACC CCC TGG AC | Common | A | |||
| 37340 | GGT TCA GAA GGA GCC TGA AG | Mutant Forward | A | |||
| 37341 | Fluorophore-1 | AGT CCC TTT CAC CCT GGG | Quencher-1 | MUT Probe | ||
| 37342 | Fluorophore-2 | CTC CTG CTG CCC AGC TAC | Quencher-2 | WT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 37338 | 0.40 uM |
| 37339 | 0.40 uM |
| 37340 | 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.