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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
>chr7:12417171-12417289 119bp ACCTTTGTGATGTGCTTTCC CTGCAGGCTCACAAGTTACG |
Mutant= 84 bp
Wild Type = 119 bp
Sequence
Wt Sequence (deletions in lower case, 3bp insertion in parentheses)
ACCTTTGTGATGTGCTTTCCCTCTCCAGCA(att)ttacttcattagcatctcaacatgtatttttctgatttcaggttcctgtactggaatgggggatgaagacgtaacttgtgagcctgcagaagaggtggtgtgtaacacaagtccatctccccaggagactcatctccttgagatacctgtcccagctttggaagaggcttggcaccagaatgagttcttgagcacacagcctgtgcagacattagatttgagcagaacaggttccggtttgaaggaagacagtaaccggcattggaagagttacagtggtgaaaagtcctgtgaaagggattctcacagggctacagctatgtcaagttgcagatcccatgggttgactgagccattcatccgtgggaaggatggtgaggaggccctctcctctatagctggtgttcagcagcagggtgcgtgtcatagtgaacagccctccacaggctgtcttcaacagcaagccacttgtattcgtgaacagccttatagcagtgccaagtgtgggcaagcacttttgaatggagagaaacattgtaaggacggtgaatgccaaaaaggttcaagctgcaaacacacacttgttcagtgtccaagtgcctcctctgagaaaaggccatctgagtgtagcaagtatgagaaagccttctcctgcagttacaagcatgctcagcatcaggacagtcactcgggaaaaaggacacatgagcatagtcagcatgagaagcccttcagcaaaaagtgccgcctcaacacacaagacagaatttgtcctcaagaaaggccctataaatgtactgaatgtggaaaagcctttatctacaaatctgaactcgtttaccatcagagacatcacagtgctagagtacattatgagtgtgtggaatgtagaaaatccttcacgtacaaatccaacctcactgaacaccagagaatccacactggagaaaggccttaccagtgtgaacaatgtggaaaatccttcagacaaaactctagcctctttagacaccaccgaattcatactggagaaaggccatacgagtgctccgagtgtgggaaatcctttagacagatctttaatctcatcagacacaggcgagtccacactggagaaatgcctcaccagtgtgctgattgtggaaaagcctttagctgcaaagctgaactcagtcaacacgagcgaattcacagcggggaaaagccttatgagtgtagcgagtgtggaaaatacttcaggcagttctccaacctcattcgacaccggagagtacacacaggtgacaggccttacaagtgcagtgaatgtgagaaatccttcagtcgcaaattcatcctcgtccagcaccaaagagttcacactggagaaaggccttacaaatgtggggaatgcggcacatccttcacccgaaaatctgacctcatccagcaccaaagaattcacactggcacgagaccttatgagtgtgaagagtgtgggaagtcattcaggcaatgctctagcctcatccagcaccggagagttcataccggagagaagccttatgagtgtatcgaatgtgggaaagcctttagtcagagtgctagcctcattcaacaccagagactgcacactggagaaaggccctatgagtgtagtgaatgcgggaagtccttcagccagagtgctagcctgattcaacaccagagaagccacagtggagaaaaaccttttgaatgtagtgattgcagcaaacccttcactcataagtctgacctgattcagcaccgaagggttcatactggagaaagaccttacgagtgccatgattgtgagaaatcctttagccgtcgatcgaatcttgtccgacaccagcgagttcacacctaagaagagtcttcatcatgtaacatgctgcctagcgtgttctttatcacatctaggtgagaaggacgtaggcagaaagtgcataaagatttatgcctgcccctgattggacgaaggtgggaagtgtgcagctttgtgggttttgcctttataagcagctggctaacataatttgtggccattctctgggaatcccaggtttgaccctggccagtatttaataaaactttcaaatttgactcaaaaaaaaaaaaagtgctgtttaataagtgtagtaacactggaggagactcagagccctctctgaATTCAGAGATTATTCATCTGGGCTTCAGAAGTGGAGGTTATTTCAGATATGTGGGAAATTTTGAGGAGACTCATTCCAGTTT
This mutation is a 2174 bp deletion beginning at Chromosome 7 position 12,415,086 bp and ending after 12,417,259 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 62769 | ACC TTT GTG ATG TGC TTT CC | Common | A | |||
| 62770 | CTG CAG GCT CAC AAG TTA CG | Wild type Reverse | A | |||
| 62772 | Fluorophore-1 | CTC CAG CAT TAC TTC ATT AGC ATC TC | Quencher-1 | WT Probe | ||
| 63387 | CCA CAT ATC TGA AAT AAC CTC CAC T | Mutant Reverse | A | |||
| 63743 | Fluorophore-2 | CCT CTC CAG CAA TTA TTC AGA GAT TAT | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 62769 | 0.40 uM |
| 62770 | 0.40 uM |
| 63387 | 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.