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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
>chr13:67593824-67593920 97bp CACAGAATGCAGAGAATATGGA TTCACTCCTGTAGGGCTTCAC
Mut= 106 bp
Wt= 97 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
GGCTTCATTGAATACAGGTGTAAAAAAATGAATGTAGCTTATCATTTATAAAGACAAGTGCAAGTAAGACAAGACAAGTGAGAAATGTTGGTTTTTAAAGAGAAACATTATAAATTTACAGAATTTTACTCTGCTTTTATTAATTTCTCAATCGTTTCTCCACAGAATGCAGAGAATATGGAAaagtttttgagtggtccaaagtgtttcaaaatcaccagataattgacttagaagtgaagccctacaggagtgaagaacgtgccaagtccttccatattccatcattaagttctgaagagagcacaattcatgcaggagaaaaaccttacaaatgtgaagtttgtggcaaggccttctgcattccattattactttctaaacacaagataattcataaaggagagaatctctacaagtgtgaagtgtgtggcaaggccttccaacatccatcaagactttccagacataagaagattcattcagaagagaaaccatacaagtgtgaagtatgtggaaaagccttccactttccatcattacttttggtacacaagagagttcatacaggagaaaaaccatacaagtgtgaagtatgtggcaaggcctttcattatccatcaatactctctaaacacaagcgaattcatacaggagagaaaccatacaagtgtgaagaatgtggcaaggccttccacatttcatcatttctttctaaacacaaaataattcatagaggagaaaaaccctacaagtgtgaggtatgtggcaaggccttccattatccatcaagactttccaaccataagaaaatccattcagtagagaaaccatacaagtgtgaaatatgtgggaaggccttccgcattttatcactcctttctaagcacaagataattcatacagaagagaatccttacaagtgtgaagtatgtggcaaggctttcgattatccctcaagactttctacccatgcaaaaatgcatacaggagagaaaccatacaagtgtgaagtatgtgaaaaggctttccgttctctgtcatcactttctaaacacaggagaattcatatgggagataattattataacagtgaattatgtagcaatgccttcgtttatccttcaagactttctaaacataagaaaatttgtagaggagagaagccctacaaatgtgaagtatgtggcaaggccttccatgtttcatcactgctttctaaacacaggacaattcatacaggagaaaaactctataagtgtgaagtatgtgggaaggccttctattatccatcaagactttccacgcataagagaattcatacaggagaaaagccatacaagtgtgaagtatgtggaaaggcgttctgttttccaccatcactatctaaacacaagagaattcacacaggagagaaaccatacaagtgtaaagaatgtgggaaggccttccgatttccatcatcactatctgcacacaagaaaattcatacgggagaaaaaccctacaagtgtgaagtttgtggcaaggccttccattatccatcattactttctaaacacaagataattcatacaggagaaaaaccctataagtgtgacatatgtgaccaggcatttcatgttccatcaaaactttctcatcacaagataattcatacaggagaaaaaccctacaagtgtgaagtatgtggcaaggccttccattattcatcactactttctaaacacaagattattcatacaggaaaaaaaccctacaagtgtgaagtgtgtgacaaggccttccattatccatcaagactttttagccatacaaaaatgcatactggagagaaaccatacaagtgtgaagtgtgtggaaaggccttctgttctccatcatcactttctaaacacaagagaattcataaagttgaaaaagcctacagttgtgagatttgtggaaaaaccttctgcATTCCATTATTACTTTCTAAACACAAGATAATTCATACAGGTGAGAATCACTATAACAGTGAGGTATGTAGCAAGTTCTTTGTTTATCCTTCAAGACTTCCTAAACATAAGAAAATTTATAAAAGAGAGAAACCATACAATTGTGATGTATGTGGGAAGGCCTTCGATTATCCATCAAGACTTTC
This mutation is a 1775 bp deletion beginning at Chromosome 13 position 67,592,123 bp and ending after 67,593,897 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 52506 | CAC AGA ATG CAG AGA ATA TGG A | Common | A | |||
| 52507 | TTC ACT CCT GTA GGG CTT CAC | Wild type Reverse | A | |||
| 52508 | ACA AAG AAC TTG CTA CAT ACC TCA C | Mutant Reverse | A | |||
| 52509 | Fluorophore-1 | AGT TTT TGA GTG GTC CAA AGT GT | Quencher-1 | WT Probe | ||
| 52510 | Fluorophore-2 | ACA CAA GAT AAT TCA TAC AGG TGA GAA | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 52506 | 0.40 uM |
| 52507 | 0.40 uM |
| 52508 | 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.