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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
>chr11:20727690-20727833 144bp TGGTTTGGTTTCTTGTTTGG CATAACAAGGCTTTTCAAGTTTC
Mut = 157 bp
Wt = 144 bp
Fam = Mut
Hex = Wt
Sequence
Wt Sequence (deletions in lower case):
CACACTAACACAGGAACCTGAGTTTTATCCACTGGAACCTTAGGAGTGAATAGGGAATAGGTGGTTCCCTAAAGCCTGCACATTCCAGACCAGTAGGACATCAGGTCTCAAAAATCAAAAAGGTGGACGGAGCTCGAGAGACAGTATGTGAATTGCACTCGGAGGTACATATGTACCTGTATACACACATGGAAATACATATACACAGAGTAGAGCAATGTAATTTTCATTGTTTTGGCGGTGGTGGTTTGGTTTCTTGTTTGGTTTTTGAGACTAATGcccaggttgactggctcaggatgaccttgatgctcctcctggcactccctgagtgaccttgccagataactttgtattgttttaaagaaacttgaaaagccttgttatgtaatctgcctttgaaaataaacaaaactaatgcttttcctttttttaaaggcataaactaactactggaaagctgcggaataatggagccagacatcattcgaatgtactcttcctcccccccacctctagacaacggagcagaggatgatgaggaggatgagtttggggagtttggtgggttttcggaagtcagcccttctggtgtagggtttgttgattttgacacaccagattatactcgtcccaaggaagactttgtaccttcaaaccactttatgccaattcatgaatattcagaagatgtagatagccttacaagctttaagtctgttcagaatggtaatgataaggatatcactgctgaactttctactcctgtgaaaagtcaatctgatgttgtactttctaccaccagcaaagaaatgattccatctaaaactttggatccttccatcgatggtatggaaagcctggaagacttagataaagtagtggtgcagggaccaagcactgggcaactcagaagtttctctcctggagattttagaactgataagaacattgttcaccaaaccaagcagttagagagctgcaacggtgaaaagcctccttgtctggaaattctaacaaacgggtttgcaggtttggaaactgtaaatcctcagggaacagatgacctggacaatgtggcagattcaaagggatcgaagcctcttaacacttgtggcaccgagtgcattttagagtctgcagctagtcatgctacggaatttgcagatttttctacattttcccaaacagaaaggacacaactggaagaaatagaatgtccagttttaaatgatggtgatacactgaccattcagggaaacagcaaaggccccagagtcaaggaactaaactgtgtgaaagaagtgactttggatggaagctttgaggatacggggaacactgaaagagaacatcaggtctgcgtctcagaaatccatgccgtagctgacagagggctcagtgttgaaaaacaagaccttcaaacactgcaacaggatgagtttctaaattcaaggattcagtctgaggcttggagcttggtagactcaagtgaaaattcagaagccattacaaaggagcggtgtaaaatggaaaaaaatgacttatttgcctctaagtgtgctgacttatccatggattctgttaaaacttcagacgttaatgaaattggttcttccaaagaagaaaacagaaagctgactaatcccaaaagcccagaccctgatcccacaggacaaaatgctttggatgattctgctgcaagcatgaaaaatggtgacagtgggaatggctttgtgacttgtcatgataccaatgaggatgattttggtgactttggtacagccaatggcaccactccaccctttgttactagtactcaagattcaatgagtgacgtcacttttgaagactcctcagagcattttctacatcttagtgaaccaggtgatgattttggagaatttgaggatacaaatgctgtttcttgtcaagaggaaatgagatttactgagtcagacctaagacagacttctgatggcttatcagaggaatgtccattggcaggagagtctggtggaaaagacagtaaacctgactcaaaactgaaaaatggtcaagacagtgaatttggagattttgattctgtgccaaatactcaaggtagtgcttttcaagactcggatgattttgcagacttcagttcagcaggtcctagccaagctgtagattggaatgcttttgaggatgaacagaaagatggttgttcttgggctgcctttggggatcagcaggagactgagtctcatcatctaaaggaagtctggcagtcacagaggacagatgaaactatgggcactctgggaacccctaagatgcacagtgtttcttcagcagcttccaaaggagcagttgctagtggccatttgcaggaaccaggcacttcagttcaggtatgtacttactgcctctgatttgtagaacatattaattgccaaggagacttctaatacagctttaaacaaaattacctgagtatctgtcaaagaggtttctgtgtgatatatgaggtttgttggtctggtttgcacattagttagatttttcacaaccctttgaaaaattgtaaaactgctacacagggagtgtcttaactagttaacaggaatttatcaatttaaattatccatggaTAGTATCTGTTTTAAATAGTAGCCATTTTTCCTTCTGTTTTTATCTTTAATAATTATTTCAATTAACAGACTGGTGAAATACTTTCAGTTTAGAATGGGTTTCTGAGCAAAAGACAACTGGGAAAACCCTTTTTTTGTTTGTTTGTTTGTTTTTTGTTTTTTTGAGACAGGGTTTCTCTGTGTAGCCCTGGCCATCCTGGAACTCACTCTGTAGACCAGGCTGGCCTCAAACTCAGAAATCTGCCTGTCTCTGCCTCCCGA
This mutation is a 2354 bp deletion beginning at Chromosome 11 position 20,725,445 bp and ending after 20,727,798 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 48889 | TGG TTT GGT TTC TTG TTT GG | Common | A | |||
| 48890 | CAT AAC AAG GCT TTT CAA GTT TC | Wild type Reverse | A | |||
| 48891 | CCC AGT TGT CTT TTG CTC AG | Mutant Reverse | A | |||
| 48892 | Fluorophore-1 | CAG GAT GAC CTT GAT GCT CC | Quencher-1 | |||
| 49709 | Fluorophore-2 | TGG TGA AAT ACT TTC AGT TTA GAA TGG G | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 48889 | 0.40 uM |
| 48890 | 0.40 uM |
| 48891 | 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.