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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
>chr16:45722784-45722870 87bp ACCTCTTCTTTTCTCTGTCC TGAGAACACACTTCCCCT
Mutant= 99 bp
Wild Type = 87 bp
Sequence
Wt Sequence (deletion in lower case):
ATCCCACCTATTTGTTGGGCATCGTAGGGCTTGGCTTCTAAGAGTCTCTGAGAAGACCCAATTCAGTTCTTTGACGCATCACAAACTTAGATAGGATGAGTTAGTGAAAACAGCAGATCCCAGAGAGCAGATCAGAATCCTCCCGGTTCCCCGTGAGCCTCCAATGCATCCTCCAttaagaatgggagatcatgtcctcatcatccaggattttcaaggagggaactaggttctggctgcaagattgtggtcagctattgtctgtgaatgggctccctgggggtcttctgtggtgtgggtgtgtgtctgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtagatgcaccactcacttctgacctgtgacatttattgcaaagggaaacacactaaccctctgtcccccacactccctttttaggtcctgtgcaagctgataaacagtttatatccaccagggcaagaacccatccccaagatctcagagtcaaagatggcttttaagcagatggagcaaatctctcagttcctgaaagctgccgaggtctatggtgtcaggaccactgacatttttcaaacagtggatctctgggaaggtaaactgccctcctggctttaggtctgtctcccctggactcttgtcccagtacctcttcttttctctgtcctctatttgcaaaagccacttacacagggattccctgtgctgcTTGGGGGAGGGGAAGTGTGTTCTCAGCTGTCATCCCAGGGACAGTGCTGGCCTTCCTGTTTGTGATGCTTTCTGGGTAGGGGTCCTGTCGTACAGAAGGACACATATTGAGGCTAGGGCAAGAACCTCAGCCAAGTAGTGGCTAAGTGTGATAGTGGAAGTGTCCACAGAGAAGAAGGGAACTGGCTACA
This mutation is a 545 bp deletion beginning at Chromosome 16 position 45,722,809 bp and ending after 45,723,353 bp (GRCm38/mm10)
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 70858 | ACC TCT TCT TTT CTC TGT CC | Wild type Forward | A | |||
| 70859 | TGA GAA CAC ACT TCC CCT | Common | A | |||
| 70860 | Fluorophore-1 | TTT GCA AAA GCC ACT TAC AC | Quencher-1 | WT Probe | ||
| 70861 | TAG TGA AAA CAG CAG ATC CC | Mutant Forward | A | |||
| 70862 | Fluorophore-2 | AGC CTC CAA TGC ATC CTC CA | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 70858 | 0.40 uM |
| 70859 | 0.40 uM |
| 70861 | 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.