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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
>chr1:54763597-54763756 160bp CCATGGTGCAGAAGTTACCTG CCAGACAGAAAGACGTGAACC
Mut= 172 bp
Wt= 160 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
GGATTAAAACCCAACAAGGCAATGGTAATTTCAGATTAAACAGTAGTCTGTAAGTATTCTGATTTTTTTTTAAACTGGTTTTCTAAACTAAAATTTTACTGGTCGGCTTTTTTGGTGAGGATGGTAATTGTTTTCATTTTAACACAACCCTTGTTGCTCTTACTGTGTCTGTTTTGTTGTCTAGCTGGTCTGGAATTCATTCTGTATCCCATGCTGTGTCTTAATTTGTAACTTGCTTGCCTTTTCACCCAAATGCTGGGATTCTGGGATTACACACTCAATACCAATACAACTTTTAAAAGGTGGTGCTACTAATATATTGTCAAACAGCCACCACCTTTGCTACATCTCATTCGAGTCTTAGTTATGAGTCATCTTTTTTCTTCCCATGAGATTTATATAATGTCCAGTACATATTAGGTACTCAATAAATGCTCATTGATTATTTTCAATGAGGCAGCTTCCCCCTCTCCTCCcgactcccagatcaggaaatactggtggtaaaaccagctaagaatgatctttttttgtgtggtgagttgtcgcattgtcttataaaatttaactatggtcttgctgctttatttgtcaaggtcacctggacgttgtggcgctgctcattaaccatggtgcagaagttacctgcaaggataagaagggctacaccccgctgcacgctgcagcctccaatgggcagatcagtgttgtcaaacatctcctgaacctgggggtggaggtaagctctgatggctgtatcgcGGTGGGTTCACGTCTTTCTGTCTGGGTTTTCTTCTGCTCTGAAGTGAAATCCTGTTTGCATTTTTATTCAAGTGGAGAAAAACTTATTTCCTTGGTTCATGTATTTATTAGTTTATGTGAGCTGATAGAGTGTGATAGGGACAAATAGCAGGAAACCAGAAACAGACTATAGACATAGAGAAGTATACAAATGTTTATATTTCATTCTTTTCCTTTTTTCTTTTTGGTTTTGGTTTTGGTTTTGCCTTTTGAGACAGAGTTTCTCTGTAACCCTAGTTGTCCTGGAACTTGCTCTGTAGACCAGGCTGGCCACATGCACTGTCACCTCCCAGCAAACATTTATATTTTGTGCAATTTGTAATAATCAGTCATTTTAAAAGGCCTGTCGGAAGCAGACCTTGGAAGTTGTGGCTCACAGCAGGAGGGAATAAATGGATGGCAGGGATTGAATAAGAAATGTTGATGGTGAGCTTGAACCATTAGCCAGTCCCTTCTCCTTCAG
This mutation is a 286 bp deletion beginning at Chromosome 1 position 54,763,622 bp and ending after 54,763,907 bp (GRCm38/mm10).In addition, there is a single base pair insertion (T) at the deletion site.
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 52995 | CCA TGG TGC AGA AGT TAC CTG | Wild type Forward | A | |||
| 52996 | CCA GAC AGA AAG ACG TGA ACC | Common | A | |||
| 52997 | CCA CCA CCT TTG CTA CAT CTC | Mutant Forward | A | |||
| 52998 | Fluorophore-1 | CTG CAC GCT GCA GCC TC | Quencher-1 | WT Probe | ||
| 52999 | Fluorophore-2 | CTT CCC CCT CTC CTC CTG GTG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 52995 | 0.40 uM |
| 52996 | 0.40 uM |
| 52997 | 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.