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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
>chr9:63525247-63525384 138bp CGTATGTAGAATCAGTCCTAGCAG CCTGAGGAAAGAAAGTTTTCCAGA
Mut= 129 bp
Wt= 138 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
ATATATTTATTATATATAGAGAGAAGTACTTTTAGAGCAGTTTAATAGCTTCTCATGAGATATCCCCCAGTAATGAGTTTTAGTGACATTTCTTTGACATCCTTATGTACGTATGTAGAATCAGTCCTAGCAGCCATGCcttcccatcactgggtggtaattgatgaagtgttagtaggtatgtactgatcacactcttacagagatcatggaatattttaattctggaaaactttctttcctcagggcatgaagattaggacaccgtctaagactaggaaatcaccatgggattatatattttctgtttacaacggctgtgtagacaggacagccccagacttcttagcattcaaatcacgcttcaaattaatctgggggagtattttttctttcctggaacaaattgagaaatttctcaaggactatgcaatatctgaagccaaaataaaagggaagagtctggtgtccctgcttccagagtttgagctgaagaacaaattaaccagaaatgatgttctggctgtgttagagaacccagttcacattcagatgctgttaaaccttccgggacagaggtacaagggccaagatggaaaggcagaagctgccaccaagatccaagccacgtggaaaagttacaaggcaagatcttctttcatttcttatcgccagaagaaatgggcctcaggtgtgatcgccctggcgtggcttttacattgccacaggacaagactaaagaggatagtgaaggaatcccgacagagacacctggagaacttccgcattcgagcccaggtatgccaggctgccagctgttaaggcaagcctctttttctaaatatttcctgcttgaacaagtgcagtaacttgtctctttatctttccttccctctccagcctccctccccctcagcattctgcatctataattctcctcaggatatgatacacaaactcagagcctggcctgcatgcctgcaaaatctgttataatttttctataaaagaaaatggtccttgttttgactatggtagttgtgttgggaagtcactgttctaactgcattaccttttctatacacacacacaacacacaagcgagcacgagcaaacacacacacacacacacacacacacacacacacacacaccttctgacactttaatttggtaggaaaaaacatcctcacttgtctatgttgctgtcaccactttgtcagaaagtatttaagtttgcagacagcCTTTTAATTAGTGCTCACAATTTCATTTATGTTTGCTTTCTCTTTTTTTCATTCATATTCTGTTAATTAAGTGAGCTGCCTCTAATCTTCCCCTGCCAGTAGCATCATGTAGCCACCTAATTAAATTAATTGGGGAAGATGTTTTAAGTAT
This mutation is a 1108 bp deletion beginning at Chromosome 9 position 63,524,247 bp and ending after 63,525,354 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 44689 | CGT ATG TAG AAT CAG TCC TAG CAG | Common | A | |||
| 44690 | CCT GAG GAA AGA AAG TTT TCC AGA | Wild type Reverse | A | |||
| 44691 | CTG GCA GGG GAA GAT TAG AG | Mutant Reverse | A | |||
| 44692 | Fluorophore-1 | CCA TCA CTG GGT GGT AAT TGA | Quencher-1 | WT Probe | ||
| 44693 | Fluorophore-2 | AGT GCT CAC AAT TTC ATT TAT GTT TG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 44689 | 0.40 uM |
| 44690 | 0.40 uM |
| 44691 | 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.