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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
>chr7:28368736-28368836 101bp GTGCTTCGTGCAAAGAGTGA ATGGGAGTCAGCACCACCT
Mut = 100 bp
Wt = 101 bp
Fam = Mut
Hex = Wt
Sequence
Wt Sequence (deletions in lower case):
GGCATTACCAACACCCCCTTTCCCAGGGCTGTGGTGTCACTCCCTAGGTCACGACATGCCAGCCATGCCAGGGTTATCAGGCTGAATAGTACGAAGCCGTGAGGAGGCAGAATTTCTGGGGTAGCCATCTGAGGCTCCTTCCTACCTGTCTGTTGCCCTCTggagtcttgtcatgggacaggggggcaggcaagccctttaccttcccctttgcccgcccacagtgagctcctggaggacttggaaggctgcagcagtgcagggggcatcgccgagtgcttcgtgcaaagagtgagtaggacagcttgaggctgggggacggacgctgggccgggattcatggggccatggggatctaggtggtgctgactcccatgtccccacagagcgaagattttgacatctatacattgtactgcatgaactacccaaggtgaggcaaagggagtccctgtttggcccccgggtattcccacgagccaatgtgccaaaccactaccccctaccctttaccgcccccaaccctgtgcccaggggcagccccttctagaccttgatagtcactggcctccttaactaccctgacctgatcatccctgaccctggccacctcctcccagccctgcctgctggagtgccagccaccactgatctatgctgtgccccccagctccctggccctgctccgagagctgtcagtgtccccgccagccactctgtggctacaggagcgtcaggcccagctccgtcactccctgcctctgcagagcttcttgctgaagcctgttcagcgcatcctgaagtaccatctcctgctgcaggtcagctgtgcccctgaggcctggcttgtggagagggggcatggttgggtctaacagccacccctggtaccctgactgtctgtggtctgcccctcccctttcatgttgctctgcctcctgtctcactcctcttttctctttttctcccccatccttctaccctgctggatgactgtcaggaactgggcaagcactgggcagagggcccagacagtggagggcgagagatggtggaggaagccattgtatccatgactgcagtggcctggtacatcaatgacatgaagcgcaagcaagagcatgctgcacgcctgcaggtggcccccggggtgggctggggcacctgagctgggggatgttgctcagtgtgtcccggtcactaacgcctctgttttggcttgtatatgtgtgccaccaacctgggcctgtctctatgcctgatgtggtcccagcaggaagtgcagcggaggctgggtggctggactggcccggagctcagcgcctttggagagctagtactggaaggcactttccgagggggaggcgggggaggccccaggcttcgtgggggtgagcgcctcctcttcttgttctcccggatgctgctggttgccaagcgtcgggggcctgaatacacctacaagggccacatctttgtgagtatagagtggggtctggctcagagagtcagaactaatgtatgttcccctgagttcccgaaaggggacagactggcttccaaggaccatatttagctgcgcccagtgacatttgacctttgattccagttactgggaaggctgagacaagaccaaaagttcaagactaacctgggctacagaaaaggttcaagaccagtcccgggcagcctgattaaaacaacattaacggctagaagtagGGCATGGTAGCTCACTGGGGAGGCAGGGGGGTTGAAGTAAAAGCCTGAGGCCAGTAAGACCGACAGGTGAGCTCAAGGCCAGCGTAGGTTATGTAGTGAGATCCTGTCTCAGGAGGGCACACTGATCTCCAGGTTCTAGGGTAACTAGGACTACAAAGAGAGCCAGACCCTGTCTCAAAACCCAGCCAAAAAAGCAACAGTGAGCGTAGCTGGTGGCCACAGGAACCAGTGTGTGAAGATGCCACAAACTTGTCCGCTGACCTCCACTGGCC
This mutation is a 1561 bp deletion beginning at Chromosome 7 position 28,367,390 bp and ending after 28,368,950 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 49136 | GTG CTT CGT GCA AAG AGT GA | Wild type Forward | A | |||
| 49137 | ATG GGA GTC AGC ACC ACC T | Wild type Reverse | A | |||
| 49138 | GGC TCC TTC CTA CCT GTC TG | Mutant Forward | A | |||
| 49139 | CTC ACC TGT CGG TCT TAC TGG | Mutant Reverse | A | |||
| 49140 | Fluorophore-1 | TGA GGC TGG GGG ACG G | Quencher-1 | WT Probe | ||
| 49141 | Fluorophore-2 | CCT CTG GCA TGG TAG CTC ACT G | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 49136 | 0.40 uM |
| 49137 | 0.40 uM |
| 49138 | 0.40 uM |
| 49139 | 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.