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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= 136 bp
Wild Type = 144 bp
>chr7:109331313-109331456 144bp CTGTGTTCCTCACCATCTCTG CTGCATTGTTGCTAACTAAAACGTC
Sequence
Sequence (deleted region in lower case)
GGCGCGGTAGAAACTTGTTCTAACCTTCACTACATTCCTCTTTCCACCCCGCAGCTTCCAGTACTGTTGGTACAGATCTACATGTTATTTTAATGCTGCTCCATTCAGGCTTTAGGGGTTTTGTGTGCTGGCCACCAGCTCTGCCTGGTTTTCCTCCACTTTCCACCCCCAGAAATGACTGCACACACGTTCAGTCTCAACTCTTCTCGCTCAGTGAGCATTCTTTATTTAGAGGCTGCCTCTCTTCCACAGAcccatggcatgctggctgttagcatatatcacatgatattgtaactctgctgagcttcctgaggtcatcgctatgtcgcttggccctctgctcctcgtgtattcaggacagtagcttatatagagtagataagacattttcacattaataaattagaattgtgagcatacttttgatatctaacacatggaacttcataactttctcttgttatgcctttaaacctgtaatgtcagaaaatgtatctcgtgatggagctttgtgaggatggagaactcaaagcagttatggatcaaagagggcacttctcagagaacgagacaaggctgataattcaaagtcttgcatcagccatcgcatatcttcataacaagggtaagacaagaactgtgttcctcaccatctctgtaacaggaatacatcactgttgtacattggtagagcttggtaatgcAAGAGGAGAACAGGTACATTTTCCATAGTTTCCCCTTTTAATTGTATTTATAGACGTTTTAGTTAGCAACAATGCAGTCATAAAATCAGACTGTCCGTGAAAACGTCATGATCATTCTTATTTTAGAAGTTTAAAATGGGCTAATATGATCAAAATTAAAGTAGAAAAATCAAAGTGAATTTTCCTAAAATATTT
454 bp deletion beginning at Chromosome 7 negative strand position 109331843 bp and ending after 109331390 bp (GRCm38/mm10)
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 37688 | TCT CAA CTC TTC TCG CTC AGT G | Mutant Forward | A | |||
| 37689 | CTG CAT TGT TGC TAA CTA AAA CGT C | Common | A | |||
| 37690 | CTG TGT TCC TCA CCA TCT CTG | Wild type Forward | A | |||
| 37691 | Fluorophore-1 | TTT AGA GGC TGC CTC TCT TCC | Quencher-1 | MUT Probe | ||
| 37692 | Fluorophore-2 | TGT TGT ACA TTG GTA GAG CTT GGT | Quencher-2 | WT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 37688 | 0.40 uM |
| 37689 | 0.40 uM |
| 37690 | 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.