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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
>chr14:56676330+56676447 118bp GTATTGGGTTTTGGGTTTCA CTACACACGCCTGAGCACAC
Mut= 119 bp
Wt= 118 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
GAAGGGCTTTCACGGTGACATTTTCATACCTGTATATAATGAATTGCAAGGTTTTTTTTGTTGTTGTTGTTGTTTTATTTGAAATAAGGTCTCACTCCATGACTGAGCTGGCCTTGAATTTATGTAGATCTGTCCCTGCCATCCTAGTGCTAAGATTAAAGGTTTACACTATCATGTAAATGTATATTCAAACATTTTAAGTAATAGATTCTTAAAAGAAAATGGAGAAACAAAGTATTGGGTTTTGGGTTTCATTTCATATGGCTTAGCGTGTCTGCAGAATAGTCTTGGTggcgggagggagcagtgggagaaacattaaataatcagctgtgtgctcaggcgtgtgtagaatatttggtttgcaaatattacagtatcaactctttatcttttattttttgttttgcattcattattctttaggagaaagagaaaaaaaatgaacccaaaggaaaataccagaaaaggtatgatttggacaaagaagaaaaagccagaaaagagccaaaggtattaaaatgtaagtacaaaaattaaaatagttccatgttcattgccattgttaaaaaaattattaatggattttatgtgtattatgtgtacaccagGGTATATATATGTGCATATGTATATCTGATATGTTGTAAAGGTTGGATGTCTCTTTTCTGAAATATTTGGGACCAAAAATGTTTCTGATTCCTCTTCCTGGTTCAATGAAATTTATATGTACATATAATGAGATTTCTTAAGAATAGGCCCAAGTTTAAATCCAAATTTTGTTTTATATGTACATTATATGCACTCCTTTGTAATACTATGATGTTTTTAGTGTTCCAGTGTTTTTGAGTCTTGCATTAAGAGTAAAATTTTCTTCTTAATACCTTCATACTGGCTTAAGTTT
This mutation is a 319 bp deletion beginning at Chromosome 14 position 56,676,388 bp and ending after 56,676,706 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 48262 | GTA TTG GGT TTT GGG TTT CA | Common | A | |||
| 48263 | CTA CAC ACG CCT GAG CAC AC | Wild type Reverse | A | |||
| 48264 | TCA GAA AAG AGA CAT CCA ACC T | Mutant Reverse | A | |||
| 48265 | Fluorophore-1 | AGC AGT GGG AGA AAC ATT AAA TAA TC | Quencher-1 | WT Probe | ||
| 48266 | Fluorophore-2 | AGT CTT GGT GGT ATA TAT ATG TGC ATA TG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 48262 | 0.40 uM |
| 48263 | 0.40 uM |
| 48264 | 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.