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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
>chr5:31532176+31532297 122bp GTTAACATATGGTTGCAGAGAATAGCC CCGCTTTCATTCATTTCACC
Mut = 137 bp
Wt = 122 bp
Fam = Mut
Hex = Wt
Sequence
Wt Sequence (deletions in lower case):
ACATACACACACACTCACAATTGATAAATAAAAGTATGTATGGAGAGATGATTGGTTTGGATGTTTTGTTCTGATTGTGCTGAGATCAAATCCAGGACCTTGCTCATGCTAGGCAAGCATTCTGTCACTAAGCAACAACCTGTGACCCCCATCTCCTGGAGACCAGGTAGGCCTTGTAGGTATGAGCTTCTTGCCTCAGTTTCTGAATGCCTAGTATTATAAGCTGTATGTATAAGTATACACAGCCAAGATTGATTTCCAGGAGGTGATccctggcagtgctcgctgtgatgttagacactgagatctttgatctgatacagtccttactggatcaagtagtaattttagacctgactgagactgttcttttcctgcaggagaagagttagaatatgaatttgatgagcaaggacacagcacttggctctgcagggtgaggtatgttcttctcgtttgtattctagctgtgatttgagaccgggtcttgttttgtggcagtcttccctcttttgcctactgagtactgggttcatgtgctctttccttgctaatgtgtatgtttgaaagtagccagtttcaagttaacatatggttgcagagaatagcccaagagtaacttcactctgcactttaggctagttccaagtgtgggccttggTAGCTTAATAGCACTGGGAGCTGGGGTGAAATGAATGAAAGCGGGTGTAAATGACTTGAAGTCCAACTGAGGCAAGAAAGAGTGAAGTTACCTGAAAGCATGAAATGCAGTCATCATGGACACTCTTGATAGCTGTTTTCATGAGAGTCAGAGCATCCTGCTGAGCTCCACCCCCTGTAAACCAATGCAGGTTACCTGTAGATGATTCAACTGGGAAACAGCTGGTGGCTGAGGCCATTCACTCAGGAAAGAAAAA
This mutation is a 391 bp deletion beginning at Chromosome 5 position 31,531,863 bp and ending after 31,532,253 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 48916 | CCG CTT TCA TTC ATT TCA CC | Common | A | |||
| 48917 | GTT AAC ATA TGG TTG CAG AGA ATA GCC | Wild type Forward | A | |||
| 48918 | GGT ATG AGC TTC TTG CCT CA | Mutant Forward | A | |||
| 48919 | Fluorophore-1 | CTC TGC ACT TTA GGC TAG TTC CA | Quencher-1 | WT Probe | ||
| 48920 | Fluorophore-2 | CAG GAG GTG ATT TAG CTT AAT AGC AC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 48916 | 0.40 uM |
| 48917 | 0.40 uM |
| 48918 | 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.