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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
>chr7:4924144+4924235 92bp GCGATGTGTGTGGAGTCCT ACGGTGCCATCGAGAGACA
Mut = 92 bp
Wt = 92 bp
Fam = Mut
Hex = Wt
Sequence
Wt Sequence (deletions in lower case):
TCGGGCAAACCGCATTCATTCGCTCAGCCCTTAACACCCAATGGGCCAGACTGGGACCACCCCATCCGCTGGCTTCTCTTCTCACTTGAGAAGAGAAATCCTTCCTCGGAAGTTCTTATGTGATGAATGGCTGAGCCTCTACTCCCACCCACAGGCTGGTGTAAAAgacacggaaaaccgtgtggccctccacgctctgacacgacctccagcccttctcctcctggctgcagccagcagcggactgcgcttcatcctggcctccttcgccctggccctcctgctgcccgtgtttctggctgtagccgctgtgaagctgggtttgcgagcccgatggggctctctgcctccaccaggcggcctgggagggccctgggtggctgtccgtggctccggcgatgtgtgtggagtcctggctctggcccctggtgccaacgtgggggatggagcccgagtcacccgcctctctgtctctcgatggcaccgtcgcaggggcgtgggcagaaggctgctggcctttgccgaggctcgagcccgagcatgggctggaagtatgggggagcctcgggcccggctcgtggtcccagtggctgtagccgcttggggggtggcagggttattggaggcctgtggctaccaggcagagggaggctggggctgcatgggctatatgctggttagggaatTCAGCAAAGACCTGTGATTAGAGACCATGCCCACTGGATAGATGGGCAGAGAACCAGCACCTAAAGAACACCTACTGTGTGCCAGGCACTTTACATCCCCCCCTCAGTGTCTACCTAGTCTGCTGGGCTGATAGTGAAGCACCGAGGTTGATGGACTTACATT
This mutation is a 510 bp deletion beginning at Chromosome 7 position 4,923,920 bp and ending after 4,924,429 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 49287 | GCG ATG TGT GTG GAG TCC T | Wild type Forward | A | |||
| 49288 | ACG GTG CCA TCG AGA GAC A | Wild type Reverse | A | |||
| 49289 | AGC CTC TAC TCC CAC CCA CA | Mutant Forward | A | |||
| 49290 | TGC TGG TTC TCT GCC CAT C | Mutant Reverse | A | |||
| 49291 | Fluorophore-1 | TGG TGC CAA CGT GGG G | Quencher-1 | WT Probe | ||
| 50973 | Fluorophore-2 | ATT AGA GAC CAT GCC CAC TGG A | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 49287 | 0.40 uM |
| 49288 | 0.40 uM |
| 49289 | 0.40 uM |
| 49290 | 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.