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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
>chr1:106995614+106995732 119bp ATGGCTTGCATCTGACTTGG TCTGGTTCAGAAGGATCTGACT
Mut= 86 bp
Wt= 119 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletions in lower case):
CAGAAATATCCAAGTTTTAGTGGAGAAGGATTATCTGGGGAATTATTTACCCTTGTCAAATATTTAGCATCTATAAATAGATAAAACAAATGCAGGAAACACTTAGTATGCAGTTACATTTCAGACCCAAAGTGCAAAATGGATCAGAAAAGACAGATCTGTGGTATTGGGGCTGGTGAAGAGAGCGGCTTTATGCAGCTGAGATTGGTTATGGCTTGCATCTGACTTGGCAGAAAGTCAAATGGCAGTGGGGACCAATGgacatctctattcttccttcagatttgttccccagaggaaggattgtagtcagatccttctgaaccagaatctagacttgcctctgcccagataacccactgaggtttttgtcttgattttgaagatcgagaagagagaagaaatacatcatcaactccaaatgctgttgactgaaataagcaaattttctaacgattatgacctgatcataagcaacaggctgttcggagaaaaaacatacctcttccttcaagtaagttttgcagtgtttgccacatctatatgtggtaccaagaaatggccagtatccctgttaggactgagttctggggctatgctgccatgttggctgcttgccatgggactatttcactagtcattctagtctgctttccagacaagcagttcctttcttagtcttctgagtctgtatctgtttttttctttaattgtatctgcctgagcatgcatggaagaggtaatgagaaacatttttctcggatctaaaagtccaggttttgagattagttctcctaacgaacagacacatttagttaaaaagcttttgtgcttaataagtaaatccttccatctctttctatttctttcctaaagaaatacatagattatgttgaaaaatattaccatgcatctctggagcctgttgattttgtaaatgctgcagacgaaagtcgaaagaagattaattcctgggttgaaagccaaacaaatggtagagtatgaattgggttttcaccaagaaacaccttttgaacattggctgtgaccaaagccttgggcttgatgcaacacagatgccagatggttgagctttgggagaagctcgagatgggtttggcagaattgaacagtcaagcatcagcataaagctcatctagggtgccgcagagggtagaatggcctctgCATTCGTGGAGAGATGCAACAGTGGGGAATTGTTCCCACAGGCTGTGGCTCACACTACCTTTGCAGTCTTAGGGATGGTGCCATGGTTACCTTTAGCTTATTCACTGTACCACAGACTTTCTTTCTTTCTGAGTCTGGGGCCCTCAGTGCAAATGGCATAGAGGGTTTCCACTTTCTTTATTAAGAGAAGTTCATACAACTCTTGTGCCAGGACCCATCTGCTGCCTACAGCTAGGGTCACTTGGGTCTCTTTCACACTTGGGAATCT
This mutation is a 928 bp deletion beginning at Chromosome 1 position 106,995,664 bp and ending after 106,996,591 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 64380 | ATG GCT TGC ATC TGA CTT GG | Common | A | |||
| 64381 | TCT GGT TCA GAA GGA TCT GAC T | Wild type Reverse | A | |||
| 64382 | GGA ACA ATT CCC CAC TGT TG | Mutant Reverse | A | |||
| 64383 | Fluorophore-1 | CTT CCT TCA GAT TTG TTC CCC A | Quencher-1 | WT Probe | ||
| 64384 | Fluorophore-2 | ACC AAT GCA TTC GTG GAG AGA T | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 64380 | 0.40 uM |
| 64381 | 0.40 uM |
| 64382 | 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.