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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
>chr8:61361406+61361502 97bp CCATCTCTCCAGCCCTAACA GGAACCCCACCAGACAGAAT
Mut= 110 bp
Wt= 97 bp
Fam=Mut
Hex=Wt
Sequence
WT Sequence: (deleted region in lower case)
AGATCAGAAGGTAACAATTGCTCCATTGATTGTCACAGACACAGAGCCCCGTGGACAGAACCCCATTAGACTATGCCTGCCCATAAAGGACCCAGTGACTTCTCCCTGGTATATGTGTTAAAGATTACCAGCCTTGAGAGTCCAAAATTCCAAAAACTGGCCAAGTGAAGAAGAGGAAAAGCAAAGGAAGCCACAGTGGCCTTGCGACTATGGCTTGCTATGGTTTGGGCTCTGGGAAGACCAGGCATCAAAAGGAACAACAGCAGGAGGAATGGCTTGCCATACAGCGGTACAGCGAAGTATAATGTATTTAAGGTCCAGGTTACAAAAGCCTAGCAGAAAATCAATTCTAGCCAGCAATGCGACATTTTAGCAGTTCCTTTGGAAGGTGTCTCCATTCTTGTCCAAATCTGTACTTCTTATTTTTAATTACTATTTATCTTGTGTGTATGAACGCTTTGCCTACATGTCTCTGTGCACCATGTGTGTGCCTGATGCCATCAGGAGCCAGAAGAAGGTGTTGAATCATGTGGTCCTAGGGTTATGGGTGGGTGGGAGCTGCCAGGTGGGTGCTAGGAATCACACCTGGGTCCTCTCGAAAGAGCAACCAGTACTCTTGACCACTGAGCCATCTCTCCAACCCCAGTTCTGTACTTTCAATGTGTTATTAATTTTAAACTTCTACACTGTCATTTAATTTTTTCCCCTTCCTGTCAAGGTTTACCTGTTTGCCAAAAAGAACGATGCGGTTTTAAAATAAACTTTTTTTTTAAAAAAGGTTCATTTATTTATTTTATGTATATGAGTACACTGTAGCTATCCACACCAGAAGATGACATCGGATCCCATTACAGATAATTGCAAGCCACCATGTGGTTGCTGGGAACTGAACTCAGGACCTCTGGAAAAGCAGCCAGTGCTCTTAACCGCTGTGCCATCTCTCCAGCCCTAACATAAATTTTTAAAAAGGGTGTTTCATGTGAAGTAACctgctggttctgttgtgcatggattctgtctggtggggttcctcattctcctttccttatacctttcataaagatataaataaatcatatctgtgtcccctccccatctttttttcccctgtattagagtatgaatcctccacttcccccaccccctctcctggcggccaccgtactcgcctccaccccgtcaggcgctactgctgctgttgctgctgctgctgccgccgccgctgctggaatgggacccaggcctgtgatggggtcctctgaacagattgcacatttacggcctcagactcgtcccagtgtgtaagtgagggtgtctttattaccacgtaaacactcatctcaaacagacatcaagaagcacaagtctgatgggagctactgacaggcttagggctcaaccaagtggcttgtattctgaaaacttCTACCTGGTTATGCATATAATTAGTAAGACACTTAGAATGAGCCTAATGTGAGCCTGGTGGGTGGCTGTCCCGCTGAGAAAAGGCCTTTCGCAGTTTAGAGGCATCTCTGTTCTCTCCTTTATAGGTTGCCTACATAGAGAACTGCTGTCCTTTCATACTGCTCTGTTGTAACCGTTTTATCTTCAGTTTCATTCCTTGTATCAAGATCTTAAGCAGCAGCAGTTCTCAACCTGTGGGTAGTACGCAACCCCTTTGGGGAGGTTGAATGACTCTTTCCCAGGGAGCGTATATTAGATTATTTACGTTACGATTCATAGCAGTAGCAAGATGACAGTAATAAAATATTTTTATGGTGGGGGGCCACTACATCAGGAGCTACATTAAATGGTTGTAACATTAGCAAGGTTGAGAACTGCTCATCTTAAAAACCAGGAAGGTATTCCACACACATCAATCAGGTCTCCCTGCAGCAAGCATCTAGAGAGCTTTAAGTCCCCATCGGTAAGGAATCCAGAGAGGTCCAGTTACCCTGTGCAGTTTGCCGCCAACAAAAGGCTTCTGCTCTCTGTGCCTCTGCACCTGTAGGGTTCAGAGCCCTGGCCGATGCAGGTCGGTCGCCTGCCTGGTCTCTCTGTGTTGACAGACACCTTGTACTCGGCAGTCCTTTTGTGCTCTCTACAAACCAGAGTTCCTTTGATTTTCTGTATCTCAGTCAGCTTGGTGACTGAGCTCAGAAGTGCAGGTGCAGCCTAAGTCT
This mutation is a 436 bp deletion beginning at Chromosome 8 negative strand position 61,361,896 bp and ending after 61,361,461 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 37882 | CCA TCT CTC CAG CCC TAA CA | Common | A | |||
| 37883 | GGA ACC CCA CCA GAC AGA AT | Wild type Reverse | A | |||
| 37884 | GGC TCA CAT TAG GCT CAT TC | Mutant Reverse | A | |||
| 37885 | Fluorophore-1 | TGC TGG TTC TGT TGT GCA T | Quencher-1 | WT Probe | ||
| 37886 | Fluorophore-2 | CTG GTT ATG CAT ATA ATT AGT AAG ACA CT | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 37882 | 0.40 uM |
| 37883 | 0.40 uM |
| 37884 | 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.