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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
>chr1:34473174+34473298 125bp TAACCCAAGGCCCCAAAGA TGCTTTTAAGAGTTCCAGGGAC
Mut= 140 bp
Wt= 125 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case and insertions with carrots ^g^ (GCACCCATTGCA insertion) ):
GGTAGAGACAAGTGAACCCATTGAGGGGTTGCCAGTCAGTCAGGCAAGCAAATCTGTGAGCAGCCTGTTTAGTAAGAGATCCTTTCTTAAATTTAATATTGACCTTTGGTCTCCACACTGTCAACACACACACACACACACACACACAAAATGGGTGCATACTAATGTACAAAAAACCCCAAAAGGTTTTATTTAAAGTTTTAATCTAGGGATCCTGCAATT^taagaggtactgatctccttcaggtcagctcagaaggggttgtggtgtctgtacagttaggcagtcctgacatcccagcacttaggaagctgaagtcagaggctagcctgggcaacctagcacaagacctgtgggaaaaggaggaggaacagaatggggaaaggggcattgtgggtaggctatgatggtgtctgctctgattcacgattgtcagtgctggctgcggacgaacaggtgtcctgtgcgctgttgactatgtgaggcagttgctgctgacccaggtatgacgtcctctgctctgtgtgcagtatgtcccatgtctgcctaccagggtctggtcacacttcccctctctatcccagacaatccctcccaacttcagtctcttccaagtggtcctggagatgcggaaacagcggcctgcagcagtgcagacagaggtatgtgcttgccccgtcttcctgttcctaaggacgagggaggtttgcttagcttcccaatctccccttctcaatgaccactcaccccaacaggagcagtacaggttcctgtaccacacagtggctcagctattctcccgcactctccaggacaccagcccccactaccagaacctcaaggaggtacagagcccccaatttcaccctttctgccagccatcaacacctatacccttcttgggaacagcaaccgacttactgtgaaaggcacctttgcctcctcagagctccttccccaggctgttccctaagctcacctaggcacccacagtgacctgcagcagggctatggcctcaggaccgaccctgcctgcttccctgcagaactgcgctccaatctgcaaggaagccttctccctcaggacctcctcagccctgcctgccacatcccggccaccaggaggggttctcaggtactgggctcactctgtccctggaaaatcaacctctacagtctgggttagcttttcccttggtcttccagaaggtaattaattaggcaagtacatttttaaagaatttcaatctctctctctctctctctctctctctctctctccctctctctctctctctctctctctctctctctgtgtgtgtgtgtgtgtgtgagagagaaagagtatgtgagtccttaccttccacgtgttttgagaaacagtctcactgtgtacatcagagcttctggggactcacctgtctctgtctcattgtaggagggttggggtctcacacactctaccacatgcgtcctccctagtcccacttgccaatctggccctgtccctcctgctgtggggattctagggacacggaacctctactttctgccctcaggtggcccgccccttccctcctttcctgctctgaagactggggcaggcaggaaggaagcccgtagcctgggctgagcttgctcccccccccccgcccccctcccttgtgctaagcgctttcacttcctctgcttgttgcccgctgcaggagcatctcggtgcctgcgcccccgaccctccccatggctgacacttacgctgtggtgcagaagcgtggcgcttcggcgggcacagggccggggccgcgggcgcccaccagcacggacaccccgatctacagccaggtggctccacgtgcccagcgaccggtggcacacacggaggacgcacaggggacaacggcactgcgccgaggtgagttgaggccctccgggggtgtggagctgaggacaagtacctattagcaactttcccgctctcctaaggctcccgagggtctaagcgctgctcggaggtcagccccagatccagcatggccacgcccctaggctactaaacgtcggggttcaagccccaaaatgagccccaggtcaggtctcaattgggagactgggctgtctaacccaaggccccaaagacagacctccgaacttggacacgcccatggacacgcctcctaccgctacctcgggaagg^GAAATCGGTAGCTATGCTGCTCTAGAGTCCCTGGAACTCTTAAAAGCAGCTTGAGCTGACCCATAGGGGTTCTCCTGCCCTCAGTTCCTGCGGACCAAAACTCTTCCGGGCCTGATGCCTACGAAGAAGTAACAGATGGAGCACAGACTGGAGGGCTAGGTAAGTCAGAGCCTAGGGTGCAGCTTTGGAGAGCCCCAGGACTTCGGGAGTTCGGGCTGGGGC
This mutation is a 1972 bp deletion beginning at Chromosome 1 position 34,471,279 bp and ending after 34,473,250 bp (GRCm38/mm10). In addition, there is a 12 bp (GCACCCATTGCA) insertion at the deletion site
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 52772 | TAA CCC AAG GCC CCA AAG A | Wild type Forward | A | |||
| 52773 | TGC TTT TAA GAG TTC CAG GGA C | Common | A | |||
| 52774 | CAC ACA AAA TGG GTG CAT ACT | Mutant Forward | A | |||
| 52775 | Fluorophore-1 | AGA CCT CCG AAC TTG GAC ACG | Quencher-1 | WT Probe | ||
| 52777 | Fluorophore-2 | CTG CAA TTG CAC CCA TTG C | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 52772 | 0.40 uM |
| 52773 | 0.40 uM |
| 52774 | 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.