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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
Mutant= 108 bp
Wild Type = 149 bp
>chr1:44114942-44115090 149bp AGCATGTATACCACCCATTG TCACTTAACACCTGAATCACA
Sequence
Wt Sequence (deletion in lower case):
AGGTATCACAGAGGCTTTTGGGTCCATTATGCCTGAAATTATAGATCATTATAGGTCCTAGACACATAACTCCAGTTCTCTGCCAGAGCAACAAGTACTCTTAACTGCCAAGCTATATCTCCAGTCCCATGATGATTATGTGAAGTATAAAGACAAATGAATTACATCTACATAGAGCATGTATACCACCCATTGACTTGATTTTCACCTATGCAAAGAGggccagattggaaaaatatagggagaagagctgtggcgacttgactagctagaaagttgatagttcatggaagatttctttattgtgattcaggtgttaagtgagatgttcaccgctgtgttatactgctgtctatctgtaaataggaccagtttaccatgagaactgtgactgtcctttggaagacagtgcagcctggctacgggagatgaactgttcggaaacaatctctcagattcagaaagatctggcacatttccctactgttgatcctgaaaagattgcagcagaaatcccaaaacgatttgggcagaggcaaagcctgtgtcactataccttaaaggacaataaggtataatatgattgaggtttgcttgttctctggactctgtaatatcttgaccttttgattatatctggctactttttttaaacatcattctaggtacatattttgacaagcctttttttttccatatctgccagtccctttgcacacatgtagtaaatgtcaaattctcttctaggtttatatcaagactcatggtgagcacgttggctttaggattttcatggatgcaatactactttcattgactagaaaggttagtatattttctctgcattatgatagcaatgtcaatctagGTTTTATAATTTAGTTTTTCAAAACATTGTTAACATCAATATAAAGTGTTCTATTCTGGTGCCTTCTATATTTGCTATTCTAAAGTTTATTTTTTTCCTAATATATGTATTAGATACTAAGGACAGCTATATATAGTACCTTTAAAAAGCAGAGGATGGCTCTGGAGACCAC
This mutation is a 648 bp deletion beginning at Chromosome 1 position 44,114,398 bp and ending after 44,115,045 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 70000 | AGC ATG TAT ACC ACC CAT TG | Common | A | |||
| 70001 | TCA CTT AAC ACC TGA ATC ACA | Wild type Reverse | A | |||
| 70002 | GGC ACC AGA ATA GAA CAC TT | Mutant Reverse | A | |||
| 70003 | Fluorophore-1 | ATA TAG GGA GAA GAG CTG TG | Quencher-1 | WT Probe | ||
| 70004 | Fluorophore-2 | CAC CTA TGC AAA GAG GTT TTA TAA TTT AG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 70000 | 0.40 uM |
| 70001 | 0.40 uM |
| 70002 | 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.