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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
>chr7:109557340-109557466 127bp CTTAGGTCTCAGTCAGTCTCTCCAC TGAGGAGCACCTGGGATTTAG
Mut = 145 bp
Wt = 127 bp
Fam = Mut
Hex = Wt
Sequence
Wt Sequence (deletion in lower case):
TTACTCAGGTCTCTGAGAATGAGCGAGACAGAAAGGTTCGTGGAGGTTGGCCTGCCGTGACTAAAGAGCTATTCTTTACTTGAGGACAGCCTTAAAGATACATATTAAATGAAGGATTTCTTTTCTTTGTTTTGGACATACTAACTCAAGCTGGCCTCAGACTCACTGTGTAGCCAGTGATGACCTTGAACTCACAATCCTCTTGCCTTCATCTCCCAAGTGATGGGATTGGTATGGGAGGGGGCACTTAGAGTATTTTTGAGACTTCTGCAGGAACATAACATCAGAATGAAACACAGTCCCCCTGGTTTTTAGACCACTCACCCTTTTCCTTTTTCCTTTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCTCTTAGGTCTCAGTCAGTCTCTCCACCTCCAGTTTTATACCCACCAagaagtcccatctacccactcagtgacagtgaaacctcagcctgcaggtaccccagccactctaaatcccaggtgctcctcaaggaccggcattctcgaaatccttcactcctaggtcaagatccgtccccagaaacctcaccacccatttgtaccctcaaggctaccagcttcagctatttggacagaaccccttcattacgcaaaagagaggaccagaaggagactgtccagggtgcagtccaggatgtagaaggtgtggctgcttgcctcccccttgcccagagcaccccattcctaggggccgggtctcggagtgtcttgctgagttgcactggtacccgagcccatagcctgggtatccgagagaagatctcagcatgggaaggtcgccgagaggcttcacccaggatgagtctgtgtggagagaagcgggagggccctgggagcgagtggtcggtcagtgagggctgccccagtgtgggctgccccagtgtggtgccatccccctgcagctcagaaaagacctttgatttcaaaggcctccggaggatgagcaggaccttctctgagtgttcctaccccgagacagaggaggaggcagaggcacttcctggacgggactctttataccggctggagaagcgtccaggccggactgagcccagtgccttgctcagggggcatggcatcaggaaagagagctcagcagtgctgagccggatccagaagatcgaacaagccctaaaggagcagcctggccggggtctcccccagctccctagcagctgctacagtgtagaccaagggaggaggaagactggaactttgggcaccttggaggagccaacaggaactgcaagtgtaagccccagcagcagggcaggaggagtggccggagttgctggggaggcaggcccacccctggacagggaaggcagtgcttccatgaagtcagagacccctggaaatagctccagtccccagctgctgcctccgaagagctcccctgatccagctgtgaaccctgttcccaaacccaagcgtacctttgagtacgaggctgacaagaatcctaagactaagccaagcaatggtctacctccttcacccacacctgctgctccaccccccttgccttccaccccagccccaccagtcacccggagacccaagaaggacatgcgtggtcaccggaagtcacagaacaggtaaggCATGTGCTCCTGTGTCAGGAAGCAGGACCTAATGGCATTCTTTTAAATGGAAGAAGCAAGAGCAGCATGTGCTTGGGTCACAGGCGTATGCTACAAGCTCTGAAGCGCTGAGTAATTAATTGTTAGTTTGCCAGTTTGGGCAGAGAAATGGTTCTCCTGCTCCCCACTACCACACACATACCTCCTTTCATTTTTGTGCCTGAACTGGAACCTGCTATAGACCAGGCTGGCCTTAAACTTTTGCCTTGGCCTCAGGGCTGGGTTTAAAGTCATGTACCACCACCAGGCCTGTCCTCCAGCTCTTGAGTGGAAGAAAGGATGAAAGCCACTCTTGCCAGTGCTGGGAGGCTTTGTTAGTGTAGAGCCAGCTATGCTAACAATTC
This mutation is a 1,217 bp deletion beginning at Chromosome 7 position 109,556,205 bp and ending after 109,557,421 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 48524 | CTT AGG TCT CAG TCA GTC TCT CCA C | Common | A | |||
| 48525 | TGA GGA GCA CCT GGG ATT TAG | Wild type Reverse | A | |||
| 48526 | GAG CTT GTA GCA TAC GCC TGT | Mutant Reverse | A | |||
| 48527 | Fluorophore-1 | TCA GTG ACA GTG AAA CCT CAG C | Quencher-1 | WT Probe | ||
| 48528 | Fluorophore-2 | CAC CAC ATG TGC TCC TGT GT | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 48524 | 0.40 uM |
| 48525 | 0.40 uM |
| 48526 | 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.