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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= 92 bp
Wild Type = 120 bp
chr18:65625246+65625365 120bp GTCTCAAGTGTTTGGAGTGTAATG CCGAAACACCGAAATGTAAATG
The common primer (*59077) anneals over the nucleotide sequence containing mouse genomic variation rs244015298.
Sequence
Wt Sequence (deletions in lower case):
GAGAAAGGCGTGGTGATGCAGTGCTCCCACCTGATCCTAAAGCCGGTCCCGGCAGACCAGATGATAGTTCCTCCATCCAGCAATACTGCTGCTTCCACTCTGCAGAGCTCTGTGGGAGCTGCCACACACACTGTCCCAAAAGTCCAGCCTGGCATAGCCGGGGCAGTTATCTCAGCTCCGGCAAGCACACCCATGAGCCCAGCCATGCCCCTAGacgaagacccctccaagctctgtagacacagtctcaagtgtttggagtgtaatgaagtcttccaggatgagccgtccctggccacacatttccagcacgctgcagacACCAGTGGACAAGTAGAGTATCATTTACATTTCGGTGTTTCGGGGACGAGGCTGGAGCCGCGAGTGCTTTGACAAATGTCTTGAATTACGGTGGAGAGGGGTGTGTGTGTGTGTGTTTGTGTGTGTGTGTGTGTTCAGGCAAGCACAGGAAAGGGAAAAGATGGTTTTCATAGTATAGGTGCAGTGCTGAAATACAAGGGTAATTCTTCAGCCAGATAGAAAAGTGTGTGCTCTGGTTGGATTATGTTAGGTAATTGAGTAGGCATTGGAAGTCTGATTCTTAGCAGTAAATTCAGTGTCCTCCCATCATATGGAGCGT
This is a 107 bp deletion beginning at Chromosome 18 position 65,758,287 bp and ending after 65,758,393 bp (GRCm39/mm39).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 59073 | GTC TCA AGT GTT TGG AGT GTA ATG | Wild type Forward | A | |||
| 59074 | Fluorophore-1 | ACA TTT CCA GCA CGC TGC AG | Quencher-1 | WT Probe | ||
| 59077 | CCG AAA CAC CGA AAT GTA AAT G | Common | A | |||
| 59078 | Fluorophore-2 | AGC CAT GCC CCT AGA CCA GTG | Quencher-2 | MUT Probe | ||
| 59079 | GTT ATC TCA GCT CCG GCA AG | Mutant Forward | A |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 59073 | 0.40 uM |
| 59077 | 0.40 uM |
| 59079 | 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.