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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
>chr19:44536065-44536156 92bp AATGTGACCCACGTCCTTTC GGATTAAAGTCCAAGGCTCTG
Mut = 132 bp
Wt = 157 bp
Fam = Mut
Hex = Wt
Sequence
Wt Sequence (deletions in lower case):
AGTCAGTGCTCTTAATCGATGAGCCATCTCTCCAGCCTGGAGTATTTTTTTTTTCTTTCCTAAAGAAAGGGCAAGTCTGGGCAGGGGAGACTTCCCTTCTGGGTCTGGAGCAAGAAGAAAATAGACAAGCAGATACAGGCTTGGGCTGGGGAAGCCTGCATGATAGGAGAagctggcgaagatgtctgtcctctgcataatgacatgtgcctgcctcggtgctgagactgattctttgcctttgtaggtttcacaagctaatatggggaagctttggcagtgggcttctggaaaactccggggttattgctggtggaggagacaacggcacactgactctatacaatgtgacccacgtcctttcttcagggaaggagcccctgattgcccagaaacagaagcacactggagctgtcagagccttggactttaatcctttccaggtactgtgtcttagctgaccagacctggtcaaggcatgacgacgcatgtctgaatggaagcattctcttgtggccttcaggctgtgcctctgaactgtaatgggagaggccctgggtctaccaaatgcaagcctcctctttctccttttctgaggtgggagtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtttgggggtaggtctttagaaatatcttttattcacacagggcaatctcctggcctcaggagccagtgactctgagatcttcatttgggatttgaaccacctgagtgtcccaatgacacctggacccaagtcacaggtgaggggctccctcctccctctgacctggggctgcctcttagggtgtacagccaggttttcccaggtaaagtttggaattgtggagccagccgagtaacagagatttccagctttaccactggagggcaataaaaccccacagatggacaggtcacctagtagtcaggagAGAGGGCAGAGCAGGTGGTTCACGGTGGTCAGTGCTGTTCAAAGAATTCCTGGAAAGAAGTGAAGGGCAAGGGGGGACAGAAGACACCAAGACAGGCAAGGAGTGATATGTATCATAGT
This mutation is a 697 bp deletion beginning at Chromosome 19 position 44,535,584 bp and ending after 44,536,280 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 49662 | AAT GTG ACC CAC GTC CTT TC | Wild type Forward | A | |||
| 49666 | Fluorophore-1 | AGG GAA GGA GCC CCT GAT TG | Quencher-1 | WT Probe | ||
| 51671 | GGA GCA AGA AGA AAA TAG ACA AGC A | Mutant Forward | A | |||
| 51672 | Fluorophore-2 | ACA GGC TTG GGC TGG GGA A | Quencher-2 | MUT Probe | ||
| 52321 | TCC ATT CAG ACA TGC GTC GT | Wild type Reverse | A | |||
| 52322 | ACT GAC CAC CGT GAA CCA C | Mutant Reverse | A |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 49662 | 0.40 uM |
| 51671 | 0.40 uM |
| 52321 | 0.40 uM |
| 52322 | 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.