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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
>chr6:133036136-133036234 99bp AGGCAAGCCTCTCTTTCTGTG AATGTGGAAAAGGATCCCTGA
Mut= 109 bp
Wt= 99 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
GTTCTGAAAGTGATTATGTTAAGAATCAGACACAGCCGTCTAGAAGATTGTATTAACACATCTTTGGTAGTTCAGAAGAAATTAGATCATCATGGTGTTGACAATAAGGGCTATTTTATGGGTAACATTGATAACTATTATAAGTCTGGAGTTTATcataggaattttaggaaatgtattcatagctctcgtgaacatcatagactgggttaaaagaggaaagatctctgcagtggataagacctatatggccctggccatctccaggactgcttttttattgtcactaatcacagggttcttggtatcattattggacccagctttattgggaatgagaacgatggtaaggctccttactatttcctggatggtgaccaatcatttcagtgtctggtttgcaacatgcctcagtatcttttattttctcaagatagctaatttctcaaattctattttccttgttctcaaatgggaagctaaaaaagtggtatcagtgacattggtggtatctgtgataatcttgatcatgaacattatagtcataaacaaattcactgacagacttcaagtaaacacactccagaactgtagtacaagtaacactttaaaagattatgggctctttttattcattagcactgggtttacactcaccccattcgctgtgtctttgacaatgtttcttctgctcatcttctccctgtggagacatctgaagaatatgtgtcacagtgccacaggctccagagatgtcagcacagtggcccacataaaaggcttgcaaactgtggtaaccttcctgttactatatactgcttttgttatgtcacttctttcagagtctttgaatattaacattcaacatacaaatcttctttctcattttttacggagtataggagtagcttttcccacaggccactcctgtgtactgattcttggaaacagtaagctgaggcaagcctctctttctgtgatattgtggctgaggtataagtacaaacatatagagaaTTGGGGCCCCTAAATCATATCAGGGATCCTTTTCCACATTCTAGAAAAAAATCAGTTAATAAGAACAGGAATTTAGGAAGGAATCTGAAATTATGAATCTCATAGGCCATGAACCTTCAGACAAAGGATTCATTAGAGAGATAGAGAGAGAACATTG
This mutation is a 861 bp bp deletion beginning at Chromosome 6 position 133,036,176 bp and ending after 133,037,036 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 43578 | AGG CAA GCC TCT CTT TCT GTG | Wild type Forward | A | |||
| 43579 | AAT GTG GAA AAG GAT CCC TGA | Common | A | |||
| 43580 | CAT CAT GGT GTT GAC AAT AAG G | Mutant Forward | A | |||
| 43581 | Fluorophore-1 | TGT GGC TGA GGT ATA AGT ACA AAC ATA | Quencher-1 | WT Probe | ||
| 43582 | Fluorophore-2 | AAG TCT GGA GTT TAT TTG GGG C | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 43578 | 0.40 uM |
| 43579 | 0.40 uM |
| 43580 | 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.