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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= 90 bp
Wild Type = 107 bp
chr16:87491478-87491584 107bp TGACAAACACAGTTCAGCACAG GAGACGTGTACGTGGAGCAAC
Sequence
Wt Sequence (deletions in lower case): deletion is too big and it wont let me copy all of it here.
ACAGTTGCAAATTCTTTATTTTCTAAGTTAGGTATAATTGTGGCACTTTTGTGGAACTGTCTGTAATGCATGGCTGTGAGACGGGTGCTTTGAAATGATTTCATTTGTACTGGTGAGTTTGTGCTGACAAACACAGTTCAGCACAGTTAAACTGTGGGACGAGCTGGTCAGatggcatttgcctgccatcaaggaacttactgactttatgttgctccacgtacacgtctctttcacttgctaaccctttctatcaggtgcagcatcctgctgcaaagatgatagtgatggcctctcatatgcaagaacaagaggttggtgatggcacaaacttcgttctggtgttcgctggggctcttctggaactagctgaagaacttctgaggattggcctgtcagtatcagaggcaagtattcatcttgtactttaattatatttgagtacttgtgtggttttgcttaaagcttaaaattcaggtctagatgggtaacagcactcgtgggcaagcacagggacccgagtagtacccggttatacctgtgatcccacatggaggagtgaagccaggagtatcactggaacctgctcaccagattgagcaggacacctgagagccactgtgcatgtgcgtgcctggacagtcacatgggcttacggtgtacatatatattgtacctattccaatatgtgtacttttaccatatacaagtaaatgattttaaaataaactttaaatctactagtaataacattgagtagtttccattttaggcattggccatatgcttcgtgtgaatttgtcctcaaaagattcctattgctaagtgttcttaaagccaaggtcctctgagtgacaagtgttagagtaagggctgctatttgtgttgtcatcagagccgtgtagtccatagctattggctctgcatgcatttctgcaagcacttgccatcttatgtgaa...tatcatgttggtgaggtaagagctacattcttgtaaacaacactataacttgttcttggtgctttgtacattcagtaaactgagaacaattgataaagggaaaatgaagatcaaacagcaaaaggatttatataggagggagaagtgaactttacataagcagtggggtccaaggagcggtaaggagatcttacttgagtaagGAGGAGAACGCATTCTGGGCAAGAGGAATAGCTTAAGGGTTAAGATGTAAACTTAATGAACTGCTTATCAGCTATAGTATGGATCAAGAAAAATGCTTTTCACTGAGGAAAACGCTT
This is a 4192 bp deletion beginning at Chromosome 16 position 87,284,234 bp and ending after 87,288,425 bp (GRCm39/mm39).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 59544 | TGA CAA ACA CAG TTC AGC ACA G | Common | A | |||
| 59545 | GAG ACG TGT ACG TGG AGC AAC | Wild type Reverse | A | |||
| 59546 | Fluorophore-1 | TGC CTG CCA TCA AGG AAC TTA CTG | Quencher-1 | WT Probe | ||
| 59547 | TTA ACC CTT AAG CTA TTC CTC TTG C | Mutant Reverse | A | |||
| 59548 | Fluorophore-2 | AGC TGG TCA GGA GGA GAA CGC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 59544 | 0.40 uM |
| 59545 | 0.40 uM |
| 59547 | 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.