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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
>chr1:33747215-33747334 120bp TTCTGATGAATATGCAGTGTCC TGTGTCCACAGAAGGCAGAG
Mutant= 134 bp
Wild Type = 120 bp
Sequence
Wt Sequence (deletions in lower case):
GGTGGTATATTCTGATGAATATGCAGTGTCCCCGCCCCCGCCGTGAGCCCTCCTCTGGCTAACctctgctcgctctgaagttgcttggctgcatcccagctgcccatgtctctgccttctgtggacacatagactcaggatgatctctggccctaactgtgtgaagacttttcctcagttatgctgatatgccgtagtacttttggcttttacctttagaaaaatttacagaagtgcttacagaaactaaagatagcgcaagtgcatgctgtggcgttgctgacgtgggaagtttctgcctcatactgactgatgtttcgtgtttaggagaaagagaggaactaaacctgactgccaaccgtctgatgggccggaccctcacggttgaggtctcggtggaaacgatccgaaacccccagcaggaggaatccttgaagcatgccacgaggattatagacgaggtggtcagcaagttcctagatgacctggggaatgccaagagccacttaatgtcactttacagtgcctgctcatcggaggtgccgcctgggccggtggaccagaagtttcaatcgatagtcatcggttgcgctcttgaggatcagaagaaaatcaagaggcgattggagactctgctgaggaacattgacaactccgacaaggccattaaactcctagagcatgctaaaggagctggttccaaaagcctgcagaacactgacggcaaatttaattagccttcaaattcaccggcccttacaaaagatgtagaaaagaaaactactattttcaatagctaggtctctatgttaggcataaccacttaattgatattggtaattatttcagatggggaagtattccagtgttggcagacacaggtaacacttaacactaggaatatttcagtcacctactttgaagttttggacagtgttcccatcttgaactggactctagcactgtatatttaacttctgaagatgcacatgtagtgtgtgtgtgtgtggggggagtaaaaaaagagggggagcagcaaaagtcccactgccctttccttgacttcagcacagagcatccagcctctggagcagtgcactcacatcgcagtgtgagcagagcacccagccctggagtggtgcattcacaccacagtgaacagaacacccagccctggagcagtgcactcacaccgcagtgtgaacagaacacccagcccctggaacagtgtactcacagcacagtttgaacagagcacccagcccctagagcagtgtattcacacctcggtgtgaagagaacacccagcccccggagaggtgcactcacaccagtgtgaacagatcactcagcccttgagcagtgcactcacagcacacagtgtacctagaaggataagcatatcagaacttcaccctgcatggtctccatgtttcaggcatctgttttctctatgcatatgcatctcataccatgaatttttaggtgtgcgtggaagcatgacaaatgtgaggagcccaccttcattttaccactgtgaagctccgcccccaccccagggccagtgctgaccgctctcttcatgtgctgccttgagctcacgcgtgtgcggcgggagcacttgcctaaccacttccctgtgggcagttggatggtttcaatttgttgctgtgactacaacatacttgtatctgctgagctttattctgttcttggtcttttttatataaaattccattaatgggttcatcaggccaaaggatatatacattttcatggtctagaaacatatttctaaatttttataagttatatcagtttgtactgtcttcagtaattaaaagaatccatttcgctgaacttctagcattggccattatttttctaccgtcagacacaaagccttctatttaatgggtaccttgtttcctaagaaggctggccagcgtggtcatcttCCCGAGTTCTTTCTTTGTTTTTTGTTTTTCGAGACAGGGTTTCTCTGTATAGCCCGGGCTATCCTGGAA
This is a 1895 bp deletion beginning at Chromosome 1 position 33,745,386 bp and ending after 33,747,280 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 46693 | TTC TGA TGA ATA TGC AGT GTC C | Common | A | |||
| 46694 | TGT GTC CAC AGA AGG CAG AG | Wild type Reverse | A | |||
| 46695 | TAC AGA GTG AGT TCC AGG ATA GC | Mutant Reverse | A | |||
| 46696 | Fluorophore-1 | CTC GCT CTG AAG TTG CTT GG | Quencher-1 | WT Probe | ||
| 46697 | Fluorophore-2 | CCT CTG GCT AAC CCC GAG T | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 46693 | 0.40 uM |
| 46694 | 0.40 uM |
| 46695 | 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.