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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
>chr4:62524396-62524487 92bp ATGCCTCCACCCACTCTGAC TGAGAGTCTTGCGCTTTCCT
Mutant= 109 bp
Wild Type = 92 bp
Sequence
Wt Sequence deletions in lower case; insertions with carrots ^ct^ :
GCAGCTCCCGGACGGTGTCAACATCTCCAAGGAGGCCCGAAGCGCCATCTCCCGCGCCGCCAGTGTCTTCGTTTTGTATGCCACATCCTGGTGAGCTGAGCAGAGGCTGAAGGGAGGCGCAGCGGGATCCTGACCCCGCGTTCGTGGCCTGGAACCTACAGGCCCCAGTTCCCTGCCTCAGTAGAGAGCGACTTACGGCTCTCTTCCAGTTGTCACACATTCTGGAGCAGGTTAAACAAGACGGTTACCGTAGCCCTTTGTATGGGAAGGGGATGAACTGAGCCATGCCACACACACACACAATCCATTCATCTACGATAAATACGGCTTTCCCACCCCATTCCTGGGGGTCATGCCTCCACCCACTCTGACAGCTTCT^ct^gaagggcgttgctctttcttccagtgccaataacttcgcaatgaaaggaaagcgcaagactctcaatgccagtgacgtgctgtcagccatggaagagatggaattccagcgattcataacaccattgaaagaagctctagaaggtatccacgtactcttctgtctcttaaaccatatataaaaaaaatgctcatcagtatcagtgccattaagtcccgttcaataatacaggactggtcataactcccctctgtttagttggtaccatctcgggtatgtgataggggcctggttattccacctgtgcctcggtttgtctaattaaaccataatatcctgcttccttacagcgtacaggcgggagcagaaaggcaagaaggaggcttcggagcaaaagaagaaggacaaagacaaaaaggattccgaagagcaagacaaaagcagagaggaggaggaggaggatgaagagagactggacgaggacgaccagaacgaagaggaggaaatagacaactgaatggagagagtaggcagactgtggcgtggtggaggggaccctgagatcctgagacatgtctgtgtgaaggctttccctgcaaggcagagccatcttcggcactggtgcctgagatcaaaataaaagctgaccagaaagatcaaaaagaactcttcccaggctcagagcatctgagttacagaaaccggtcatgcgtcatcagtctgcaggttaggatgtttttgaataagcgattctgagtgactacatagtttggtattgcttgtctacttaaagtatatggctagctagtttctgatcccatttccctgcccctctgaataagtaataacatccatgttgcctgcagtgttccaagtcacagaagcactaaaggctcagggggaaacaatggggctttgagcctggtcacccagtttgactgtacagtagttgaccataggaactcgtgcttagaatggctgctctctaggagatgactaaggagctgtgagcgagcgaacgaggaaatgtcttcacctgatggtagcattacttacttggtgtctggctttctagacttacttagctttgtagtcctctagtgactgtagatgctcagtcgtctggggtggctgcactcataggacacacactctttaattctgagtgttgattagaatctgtcaaaaaaggtgccaattcttaaaagcaggggcttaatcttggaggtaactcagtgcctgccatggtaggtcctagtgctacctcctggaaggtataaaatcccagatccatctcccagacttatggctgctgcttttcattccctgggctcataacaagttccagagactttatttcttccaggcacagcctgttctgtctccatctctctggcttctgcttcccagcccggctttctcctcctcttacttatttcatcttctagtctagggactctgataacatatgtcatattccaaagtgcaaggtcctcagattccttagtcctaatctcctgccttctaaagagagttgagaacacctctgaaccttacggtaagaggaatggtgctcaggaatgtagcaccactgctgtggcttcgaacgtgtctgttggcctttctttttccccattgagtgctccaagcactttttcctgtcttagttgtgcacttgaggctgacaggtgatgtaatatagttggacactgtttactagtaatacagggaagagtttggtgagagtgactggagactgttgagaagattttaatgctttctacaaataaagaacattcttgtttaaaatgatgactcatacaatgtgcttttgCAAAGGACTCACCAAGACCCCCCTCTCTCTCCCCCTACCCTCACACATAGGCACACAGTCTTGTGTCTTGCATTACCAGCAGGTGAATGAGTATCACAAGTTAAGGGCACTGTTCTTAACCACAGCCAACATATCCTGAGATTCTCCTGGTTACTTTGCATATCTCACTAGCTCCCACTTCTGTTTGTTTGTTTGTTTTGGTTTTTTGAGACAGGGTTTCTCTCTGTAGCCCTGGCTGTCCTGGAACTCACTCTGTAGATCAGGCTGGCCTCGAATTCAGAAATTCACCTGCCTCTGCCTTCCAAATGCTGGGATTAAAGGTGTGTG
A 1834 bp deletion beginning at Chromosome 4 position 62,522,627 bp and ending after 62,524,460 bp (GRCm38/mm10). In addition, there is a 2 bp (CT) insertion at the deletion site.
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 44803 | ATG CCT CCA CCC ACT CTG AC | Common | A | |||
| 44804 | TGA GAG TCT TGC GCT TTC CT | Wild type Reverse | A | |||
| 44805 | GCT GGT AAT GCA AGA CAC AAG A | Mutant Reverse | A | |||
| 44806 | Fluorophore-1 | CCT ACC CTC ACA CAT AGG CAC | Quencher-1 | MUT Probe | ||
| 44807 | Fluorophore-2 | AGG GCG TTG CTC TTT CTT C | Quencher-2 | WT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 44803 | 0.40 uM |
| 44804 | 0.40 uM |
| 44805 | 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.