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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
>chr7:38197750+38197838 89bp CCCAGTCCTGTGTAGGCAGT TTAGACCAGCACCTCCCTCA
Mut= 104 bp
Wt= 89 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
CCTTTCCAGGCAAATGGCTTTCCCCAGAAGATGCACGTCCCTCTCGGCTTCTCCTTTATGGAGACAAATTGCTCTGGGAGCCACAGCGGGTGGGGGTGGGGGGGTGTTCCTCAGTCATGAAGGGGATCTTAGTTGGCacagttctgaggtagggagggtgtaagtgaggggcattgggaacaaccaatttgagtatcaattatgtggaccaaatggactagagaccacatcaacctttgccctgcagagtgcctggcagaagctgggcgtgctgtgacctggtgaatgtccccacttgtcatgactctcagcaagaaacaaaggccattgttggggaatgaggctgactaattgagcccttccttgttttccaggggggaccgttgggggcctgttaggtgcctggatgacgagtggacagttcaagcccgttcctcagatcttaatggagctgcccccccgctgaacagcggaaacttgttaacgaagccatggccatcatcgggaacttggactggacagacgcagtgcagctgactgctctggtcatgagcaaccaggccatgcagcagaggctcttggccatgctaacgacctatgtcaccaaggagctgcaggctgagatccgctatgaggactaggccccgccccctggggagtggggctctcattttagatgattctatggctcattgaaggtgaccagggaatgggggaagcctgacttctgggaaacttgtctaataccagcattgattgtaagccgccatgctagtgcattctgttggaccgtcctgctggagaggcatcagccatgcctcttgggctgtgtgtgctatggaagggtacaggcgtgcctgctgtaggcattcacggacagcttggagtcctcccgatgaccatgtgggtctaaaaggttctcatgttgttggctggccgaccatagcagatgcttctctttctgtgaagtgtacctccaggtacctgtcacaggatcgaatccccttagactctggggtcttggggttcagagccagctcttgtcaggattgtgctaacctgctgagtgtttccttcccttggctaaggacttcccagtggtctcatggtgacaactgtccctcctttgagtgaagccacagatgccaggcaggttctattctctgctacaggctatgccctagacgctcttgagtaactttagtttagcttgtaggagcttcttctgcctccttaagaaaaatcatggcacttcattatattttattttccatgtcgattctggactctttttgttctctccactttcgtagcctaaattcatttagctctgttttctttttcttcttactatgtttccttgggctagccttgaagtcctgggctcaagctacatagctgggactgtaggcttgtgccaccatacctagctttacctcaaagactgttgtccactctacttttcctaagccctcccgtgttctcctgtctgctggctgcaatctataattcatttgtgtgaaaagacaaaattaccaatgacttgagttcagagatcctaacatagctaggtgctatggtgcagacccatacccacggtcccagataattgaaaagctgaagggggaacatccttgggcccaggaatccaaggacaaccggacattgtggaagccgggagataggtggggacacacacatcttcttagttagcttttatttgcagtcacggaatcaagaggcagcaagtgccctggtgagacggagagagagggtggacaagggcgagcaagggaaagtggaaacagcaagaaaactagattaacctcccgtgacactggctgtttatggattgggctagcctcgcctccatttccctaagtgacggaggctcaaatctgacattagcttgtttgttttccttgtgttgtggatggaagccagggccttgcataggctaagtgccacccctgagcgctgctcctagtcctcctctctgctgattcccgcccagcctggtcccagcccttgagtgactcagctttggtctggtcggttggtcccagtgctggagctcaacatctaacattgtatccatttgattaagtatttgatacttcctccttatccctccttggaggtgctcactcactgagggcaggagctatgtgtctcggtcacttctcctgcctccagcttgctcctgcctggcaccatggcttttgtggggtagacactcaataaatatatatttttaaagctatgtatttcatatctttctgtttgtttgtttatttatttatttatatctttacttatttattttacatgttttgtctgcatttatataagtacgatgtatgtgtctggttcccacagaggccacaggaggcactggacctcctggaaacgatgttactgacaattgtgagccgccatgtgggtgctaggaactgaacccaggccctccattagggcatcaatggctaaacaatctccctcctctaggtttgggtttttaaacctgggtttctccatgtgattctggttggcctcaaactcctggtctttcttcctgcctcagccccttgaactcggagactacaggcaggcatttccaagccaagacaaatacgttttgttgttgttagaagtctttaagttcttattgtttgccttttctttgtttagttttttgtttgtgtttgtgttttgttttgttttgagacaggatctcatgtagcccaggctagcccccaactctatgtagccaaagacgaccttgaactcttgattctccagcctccatcactgccaagtgttgggaattacagagataatacctttatgtgagacttcagtaggtatttttggaagttattgttctgaaatcacttcctgtggaattcctccaggaaggaaacatgtgacaggagagacctagaatccagttttaagctttttgaaaccactgagctgctagccacggaagctggagtttggcctggtgtccctagaacccagagcctcgcttgagcaccatgtggtccctcagttttcacaaacaggtttgggacatgttttcacatgaaatagttttagtcaaatgtatgctgtctctgaagtggggtatcccaacctcagatagttccaccccaaggtatcccccatgctatatcctttcagaactactacccgactatcatcaaattactaataaattacttttccctgaaaaagtcactctctgtgtattttgtggaaaccacttctgaagcctttaacatcaaaaggaagattgctccatctgaggcttgacaggacaggtaaaagctccctggcccggttcgtggcggccatttctggccagcatgcgccatgttactgcaagccatgtttaacctccaccagccctccagccccagtcctgtgtaggcagtgctgggaccggggctctctgtgtggcggcagcccagACTTTCTGCAGTCTGAGGGAGGTGCTGGTCTAAGGGCCAGCTTTGCAGTACAGATCTCTCTGTGTAAAGGAAGCCTCCTGTGGACTTCACCCCATGGGGATAAGGCTTTGTCTTACAGTCTGCCTCAGTGA
This mutation is a 3,339 bp deletion beginning at Chromosome 7 position 38,194,467 bp and ending after 38,197,805 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 42000 | CCC AGT CCT GTG TAG GCA GT | Wild type Forward | A | |||
| 42001 | TTA GAC CAG CAC CTC CCT CA | Common | A | |||
| 42002 | AAT TGC TCT GGG AGC CAC A | Mutant Forward | A | |||
| 42003 | Fluorophore-1 | CTC TCT GTG TGG CGG CA | Quencher-1 | WT Probe | ||
| 42004 | Fluorophore-2 | ATC TTA GTT GGC ACT TTC TGC AGT | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 42000 | 0.40 uM |
| 42001 | 0.40 uM |
| 42002 | 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.