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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
>chr4:141073484-141073585 102bp ACGTGCTTTTATTGGGCTTG TCTATGTCCCCAGGAAGCAC
Mut = 116 bp
Wt = 102 bp
Fam = Mut
Hex = Wt
Sequence
Wt Sequence (deletions in lower case):
TGTAAGTGGAGTTTTTAATCTTTAAGAAAAGCAAAGGCTGGCATGGCCTTAATCCCACCTGGAGGCAGAGGCAGGTGGATCTCTGAGTTTAAGGTCAGCCTGGTCTATGTAAGCGAGTTCTGAGGCAGCCAGAGCTACATAGTGAGACCCTATGCCCCCACCCAATAAGATAGCCCTAAAAATGGGGGCTATTTGAAAGTagtctgatttggaaagccttgtcaattaagtaagaactgttgaccctgtgggcctatggcctgtagccctctgtggctctgcttttcccttggcgttgtcccttcaccaagaatagattgcatcagatcctggagtaggctttggccatctgcccagctctcctggtgtggctatatcaggaaagccctggtagcatcacatagcagcaccaggctggaggggcgccaagctcaccacccccaccctggccttacaggggagctcttcgcacaggcccccgtggaccagtttcccggcaccgcagtggagagtgtgacagactccagcaggtacttcgtcatccgaattgaagatggaaatggtacgagtgggtctgcagggctcttctgagcagaaccattacctccagaaacgggagactgatgggtgttacccccaccccctagggcgacgtgcttttattgggcttggctttggagaccgaggggacgcctttgacttcaacgtggcattacaggaccatttcaagtgagtgcttcctggggacatagactgttgtgtcctcccttgttccctggacaaactgctccctgttaagatgtccaaagatgctttctctgcTAGTGCCGCTCAGTCCTCTTCCAGGACTTCTCTGCTCTGTGGGTCCCCAAGGCTGTGTCACCTGGCCTAGCCTTCTTGGTGTCCCTTCTCTTCCTCAGCAGCCCCTGAGCTGCCAGAGTCCTTGTCAGGTCCTTTATAAT
This mutation is a 522 bp deletion beginning at Chromosome 4 position 141,073,465 bp and ending after 141,073,986 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 49656 | ACG TGC TTT TAT TGG GCT TG | Wild type Forward | A | |||
| 49657 | TCT ATG TCC CCA GGA AGC AC | Wild type Reverse | A | |||
| 49658 | CTG AGG CAG CCA GAG CTA CA | Mutant Forward | A | |||
| 49659 | GCA GAG AAG TCC TGG AAG AGG | Mutant Reverse | A | |||
| 49660 | Fluorophore-1 | TTG GAG ACC GAG GGG ACG | Quencher-1 | WT Probe | ||
| 51154 | Fluorophore-2 | CCC TAT GCC CCC ACC CAA TA | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 49656 | 0.40 uM |
| 49657 | 0.40 uM |
| 49658 | 0.40 uM |
| 49659 | 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.