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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= 115 bp
Wild Type = 143 bp
>chr14:20695347+20695489 143bp GTTGCTCTTTTACGGCAC GATTGAAGAGACGGATGCC
Sequence
Wt Sequence (deletion in lower case):
TGGCGTAGGACCTAGTCCACTCCCCACCCGCCGCAGGGCGGGGTTTCGTGCTGTCCGAGGGGGTCCGGTGTGGACATGGCTGCTCGCTGTAccgaggcggtgctggccgccctgggggtgctcagtgtctgttcggccagcagctccgggtccgaggcttccggggaggcagagagggaggagccgtgggatggcgcggttttccggcctcccgccgccctaggcgccgtggggatagcgcgcgggcccgggtccccgcctccagggaacagagaggcggtggacctaccagtgttgctgtggtggagcccaggtctgtttccgcacttcccaggtgactcggagcgcatacagtgtgcgcacggcgcgtgcgtggcgtcccgggatcgacgggcgcgcgccgacccgcggacgcgcgcgttgctcttttacggcacggacttccgcgcggctgatgcgcccctgccgcgcctggcgcaccagagctgggcgctcctgcacgaggagtcgccgctcaataacttcctactgagccacggcccgggcatccgtctcttcaatctcaccgccactttcagccgccactcggattatccgctgccgctgcagtggctgcccggggccgcctatttgcgccggcccgcgcctccgccccgggagcgcgccgagtggcgtcgccgcggatacgcgccgttgctctatttgcagtcccattgcgatgtgccttccgaccgggaccgatacgtacgcgagctcatgcgctacatccCGGTGGGTGAGAGAGCGGCGGGGCGGGGCGGGGCGGGGGTGGGCCGGGCGAACGCCCCACCCTGCTGACCGCCGTGCTCTGACTCGCTTAGGTGGATTCCTATGGCAAGTGCCTGCAGAACCGCGAGCCGCCCAC
This mutation is a 678 bp deletion of region Chr14:20,695,019-20,695,696 (GRCm38/mm10)
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 70971 | GTT GCT CTT TTA CGG CAC | Wild type Forward | A | |||
| 70972 | GAT TGA AGA GAC GGA TGC C | Wild type Reverse | A | |||
| 70975 | Fluorophore-1 | CGC TCA ATA ACT TCC TAC TG | Quencher-1 | WT Probe | ||
| 73119 | TCC GGT GTG GAC ATG G | Mutant Forward | A | |||
| 73120 | GAA TCC ACC TAA GCG AGT C | Mutant Reverse | A | |||
| 73121 | Fluorophore-2 | CTC GCT GTA CGG TGG GTG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 70971 | 0.40 uM |
| 70972 | 0.40 uM |
| 73119 | 0.40 uM |
| 73120 | 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.