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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= 111 bp
Wild Type = 136 bp
>chr12:104344449+104344584 136bp AGGGCCTAGAGTTGCTCCA TGTATTTTGGGTGCCAATGA
Sequence
Sequence (deletion in lower case)
AGTATATCAGAGACTGAAACACAAATGTACCCTTTGGGCTCCTCTTAGCATTGAGGAAGAAAGAGTGTTGCAATGAAAATTAAATTAACCATCATCAAATAGGTTAAAATGTGAGAAAGGGTAAAGTTAAGAAGGGGAGTGTCCAAAGATTAGGAAAGCTGGAATCTGATGCAGTGTGCGATTTTAAATAGAAGGCTCATTAGAAGATGTTTTAGGGTTGCCATGCTAGGGGAGAATTGTACTGTGGATGTAGGAGGAAAGCATACTAAGTCACAGAACGGCTAGGAACAAGTCAGGTTGCCTGTCCACTGTaaacactctcgggacctgttggatgattaatgcattagaaagagcaaagtcaagtcctctaggcatggataggaagaggattttctccagggagaaggctgtgtggtgaggagacccagcgacatgatggattgagggggggtcatagagtccagggactcttaccttgctcttttttattctcagccaaatagaggagctaaacctgcccaagttctccatcgctagtgactacaggctggaagaggatgtccttccagaaatggggattaaggaagtcttcacagaacaagctgacctatctgggatcacagaagccaagaaactgagtgtgtctcaggtaatacaagaagcacaggatgttttgccttgggtcctgaatgggggtggtgaagagcatgtaggctgtggggatggaacatgtgtgagaactacattgctgaggttccttaacaaatgtccatcttctacccctgtacataagctcacctaggagcccatagtggagagggcctagagttgctccatagaaccatggatactaacatgtgctgccctgcagggcaacatacagtacactgcaggggcacctacaatacactaCAGGGGCACCCACCATTCACTTCATTGGCACCCAAAATACATTGTAGGCCAAACCACTTCCATACTTTACCTCCTATACTACAGTGCTGGGACAGTACTTCTTGACCAGCACTCAGATTATTAATAGTAAAAGTAATGACAAGGAGTCATGGTGGTTTTGAGGAAAACTCCAAGAATGGGAAAGAAGATGCTTTCGACCCCTATACTACATTCTTAGGTGCACAGCCTGGCTGTAACCAATGTTCAGTCCAGCGCCCACTCAGAGCTCTGGGTACAGATCAGTCCCAGCAGGACATGGTATTTTGAGAATGTGAAACCATGGGTTGCA
603 bp deletion beginning at Chromosome 12 positive strand position 104,343,941 bp and ending after 104,344,543 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 37781 | TGT GGA TGT AGG AGG AAA GCA | Mutant Forward | A | |||
| 37782 | TGT ATT TTG GGT GCC AAT GA | Common | A | |||
| 37783 | AGG GCC TAG AGT TGC TCC A | Wild type Forward | A | |||
| 37784 | Fluorophore-1 | CGG CTA GGA ACA AGT CAG GT | Quencher-1 | MUT Probe | ||
| 37786 | Fluorophore-2 | CTA ACA TGT GCT GCC CTG C | Quencher-2 | WT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 37781 | 0.40 uM |
| 37782 | 0.40 uM |
| 37783 | 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.