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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 transgene genotype is determined by comparing ΔCt values of each unknown sample against known homozygous and hemizygous controls, using appropriate endogenous references.
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= 141 bp
Wild Type = 163 bp
>chr9:65999736+65999898 163bp AATTTGGCCACAAAGCCTCT GGAATGGTTCTGCTTCAAGG
Sequence
Wt Sequence (deleted region in lower case)
ACTCCTAGATGAGAAAAAGAAACCCCTACAGAGATTTTGTGTGAAAAGAAAACCAGTTGTGCTGGCCTAGGGCCAGGCTGCACCTCTTTGGCCTTGCCTCTCAATCCGATTCTCCAGGGGGCAGCAGTTACCAACAACTCTTTTTGGCTTTAGCAGCGAGCCCAGGGAGTGCTAATGAAACTAGGCGGATGTACCAAGGTTTGTTAGCTTGCATTTGAGCTCGGTGAGGCTCCCTTTGAACCTTTTATGATGCTGCACTTTCAGGCATCCTTTGGGAGAATAGTTTACATCCTGAAGCAATAATGGATCAGAGACCATTTTCTTTTATACAGCAGTGGAAACGCCTTTGGCACTCTGGTATTAAATAACTGCTTATTTGGACACATTGTGCAATGGCTTTGCCTTTTCCGCccagtaatagttcccacagaagctattatactcagtttaacttttccaGGAgggactctgaaaggaaattgaaatcttagagattatactttatattacattcattgctaatgtcttatgttttagtttttgtgtctctcactctgtaccccatttgcccctctactttcaggtgaaggcctcccacaggtttattactttggaccatgtgggaagtacaatgccatggtgctggaactccttggccctagcttggaagacttgtttgacctctgtgaccgaacgtttactttgaagacggtgttaatgatagccatccagttggtaagttgatgtgtggttccagaagataaatggatcccttgcccacctccttgagggccctttaatgatgtattagctgcagtgagatctggtgacttcgtttttacaggcttaatggcattctagaaggatctagcagacttctgaaaggaactattgcagctgctgctttagctggttcctgtgtataatttggccacaaagcctcttggccagtatgtaatatgccaaacagctaaggtaggttcttagccacacaacttaaagggttacagagggttaactgagggAAGTCAATGCATCTGCAGTTTGCTCTATTTTAAGAGTTTGCACCTTGAAGCAGAACCATTCCTGTAACTGAAGTGGGGGAATATAATTTACATTAGGGAGAAAGAGGACGATTAGGTAGAGCTGTGTGAATTCAAGTAATTGCTTCAGTGTCTTATTTATAGGTCTTAGGCACACAGTTGTAAGCTTTTATCATGCCTTCTTTCTCTCACTGTTGAAAACCAGAAAAGACTGAAGGTACCAACCTTTATTCAAAGTCACGTAACTGAGATGTTACAGATAATATACCATATATAACATGTAGCATATTATATACTTGTGGGCTGACCAAGAAGCTTTACAGCTAAATGAAATACTAGTTCTACTT
611 bp deletion beginning at Chromosome 9 positive strand position 65,999,223 bp for 48 bp where there is an endogenous 3 bp retention (GGA) followed by 563 bp deletion ending after 65,999,836 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 36707 | CAG TGG AAA CGC CTT TGG | Mutant Forward | A | |||
| 36708 | GGA ATG GTT CTG CTT CAA GG | Common | A | |||
| 36709 | AAT TTG GCC ACA AAG CCT CT | Wild type Forward | A | |||
| 36711 | Fluorophore-1 | AGG TAG GTT CTT AGC CAC ACA ACT | Quencher-1 | WT Probe | ||
| 37947 | Fluorophore-2 | TGT GCA ATG GCT TTG CCT TT | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 36707 | 0.40 uM |
| 36708 | 0.40 uM |
| 36709 | 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.