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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
Mutant= 123 bp
Wild Type = 122 bp
chr4:11303593+11303714 122bp ACAGAGACCCTCGATTCTGC TTATGGTTGGGATCAGCACA
Sequence
Wt Sequence (deletions in lower case and insertions with carrots ^g^ (g insertion):
CCTCCTGCCTCAGCCTCCTCTGTGCTAGGGTTGTGGGCCTATACCACCATGCCTGGCTTTTACTTTAGAGTTTGACATTCAGTTTATTGCCAGTCCCTTCTTACTGTTCctgct^aa^GTTTGTAGGGTATTCTTTGGGTTTTCCTTCTAGAATTTCCTTGTATGTAAAACATATAATGttatatcagttgtaaaccatttgaatgttttaacaaaaatgattgctaggctattgagaaatttaatcttattaatacagtagtttaggaaattttaaatatgtccttgaatacaactgtaaatgtcagtttaaaatgtgaaggaaacaaacgaaaccaaacacaagaggctggaggagaagaaagagtgcagcctgacctttactgcttgtgtcactgcaggagctcgagcgagagatcaccttccagggagacagcgctatttactactcctactataaagacatgctgaaggcgccgtcattcgagagaggtatggccagccacagcggtaactgcagcaatgatgttgttctaaaggaaagcactcttctcaaaggttatgagctttaggatgcgggatatgatagtaagttgggaagatgcagcatctactgtaacatctgagtgcctttttttccatattatttgtgattactaatgaatgacatgttgaatcactgtcggggcagaacagtaatctggagaaaaatcatgtatttaggtgtttatgagctgacgcacaataacaaaaccatctccctgaagactatcaacgccatgcaacagatgtctttgtacccagaacttattgccagtgttctctatcaagcaactggcagcaatgtatgtatttattattattattttttttattttagatttacttcatgtgtatgagtgttttgcttgcatatctatatgtgactgcatgtgtgcccagtgcccttagaggacagagagcatcagatcctctaggacactagttctcactctgtgatattctgcatatcaggtgtttacattatgattcaaaacagtagcaaaatgacagttatgaagtagcaatgaagtaagtttatggttgggatcagcacagcatgagggactgtcacagcactaggaaggctggaagccactgctccagaagttgtagacagttctgagccactgtgggggagcagaatcgagggtctctgtaagaagaaaagtgctcttagccacggagccctccctccagccctgtgtcttcactgtgacgtctgttttcctacgtgggtttgtatctttgggttgtttaaccatcctctgtcactccctttctttttactttaggaagtgatcgaaccagtgtatttctacattggcattgtgtttggattacaaggaatgtatgtcacggctttgtttgttacaagttggcttatgagtggcacgtggctagcagggatgcttactgttgcgtggttcctaattaacaggtatgcaaggtttttacattgatatttacagattttgttttggttatagagttccaaaaatatccccattcttacagtgctcagctgttatctgcaaacctattgattgttatgtttgatggtgacgattaggtaggactccacacagggactaacttagatacaggatcacttacagagccggtctaagcgcttgCTGTACAAGTATGTGAGCTGGAGTTCAGACTCCAAGAATCCACCCTTCTCAATAGTCACAAATGATATAAAATACTTTGGCGTGACTCTAACTAAGGAAGTGAAAGATCTGTATGATAAGAACTTCAAGTCTCTGAAAAAAGA
This mutation is a 1497 bp deletion beginning at Chromosome 4 position 11,303,116 bp and ending after 11,304,612 bp (GRCm38/mm10). 61 bp before the deletion site there is an indel with 2 bp insertion (AA) and 5 bp deletion (CTGCT).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 66475 | GGG TGG ATT CTT GGA GTC TG | Mutant Forward | A | |||
| 66476 | Fluorophore-1 | TGC TGT ACA AGT ATG TGA GCT GGA GT | Quencher-1 | MUT Probe | ||
| 67927 | CCC TTC TTA CTG TTC AAG TTT GTA G | Mutant Reverse | A | |||
| 67928 | TTA TGG TTG GGA TCA GCA CA | Wild type Reverse | A | |||
| 67929 | ACA GAG ACC CTC GAT TCT GC | Wild type Forward | A | |||
| 67930 | Fluorophore-2 | CTG TCA CAG CAC TAG GAA GGC | Quencher-2 | WT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 66475 | 0.40 uM |
| 67927 | 0.40 uM |
| 67928 | 0.40 uM |
| 67929 | 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.