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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= 129 bp
Wild Type = 109 bp
>chr10:100595109-100595217 109bp GGTGGCTACTCCAAGATCACA CAGCAAACCCCGAAATATCA
Sequence
Wt Sequence (deletion in lower case):
GCTTATAGCCCTGTGAAGAAGAGAGAAATAATAAAAGTATTGCATGTACGATGTGTATTATGAGGTCAAACATTTCCCAAAGTATATAATAAAAGGGAGTAAGTTTTAGCAACCTGCTTATTGAGGAAATATGAAACTAATCTTTCAAATCCTAAAGGACTAATATCATATTCTGCTCATATCTAGGACTCTTCCACCAGAGAAATATGGTGAGCGTAACAGACCAGTTCCTCCAACGAGGCTCCACTAAATatgaggtcttttgtgttatgcgaagaatctattctaagtctcaccttggaggagcattttggtatcacagcagatagctgctctgctagaactgagacacctaccccagagaatgtctgtgctctctgcacctatgatagattcctgaacttctcaaagtcttacagtaatgcatgcaaatgtgtcactctaggtggctactccaagatcacagtataataatgtatatgtccagttattacagatatactcactgctacttggtggcaaaaacaatccattgatatttcggggtttgctgtggtaaaagatacaactgatcttcacacaacccagaggttgggtttcccagaagcatgaaatgctgcagttttgctaggaaaaggaaggtgggagaaaatacatttaggcatccaataaatgcacactgatgtcacgtGACAGGAAGAGCCTTCATTTCTGACTTAAGGTCATCATGCTTTCTACATTTTGCCACCTCTAAGCTTTGCTATTTAAAAAATAAAAGCCTCTCTCTCTCCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCCCTCTCTCTCTCTCCTACATGTTTAGGTTGAGCAAAGTGAAGAACACTTAAAATTTTTG
This mutation is a a 439 bp deletion beginning at Chromosome 10 position 100,594,971 bp and ending after 100,595,409 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 67914 | GGT GGC TAC TCC AAG ATC ACA | Wild type Forward | A | |||
| 67915 | CAG CAA ACC CCG AAA TAT CA | Wild type Reverse | A | |||
| 67916 | TCT AGG ACT CTT CCA CCA GAG AA | Mutant Forward | A | |||
| 67917 | GGT GGC AAA ATG TAG AAA GCA | Mutant Reverse | A | |||
| 67918 | Fluorophore-1 | CAG ATA TAC TCA CTG CTA CTT GGT GG | Quencher-1 | WT Probe | ||
| 67919 | Fluorophore-2 | AGT TCC TCC AAC GAG GCT CCA | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 67914 | 0.40 uM |
| 67915 | 0.40 uM |
| 67916 | 0.40 uM |
| 67917 | 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.