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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
>chr8:70191963-70192066 104bp TGCATCTCTGGGTCTTGGTC CTCTCTGCAACAGAACTCCAG
Mut= 104 bp
Wt= 104 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
AACAAATTCCAGGCCAGCCATGATTGCATAGAGGGACCCCATCCCAAAACAAACAAACAAACAAACAAACAAAGTCAAGAGAAAAGGGACGGGAGATGGTTCAGTCAGTAAAAAAGTGTTTGTCAGGCATGAAGACCTGAATTCAGACCCCCAGGACCCACATGGAACCGTGACAGCCTGTGTCTGTGAACCACACTAGGGCAGGAAGGCACAGAGAAGGGAGGATCTTGGAGCTTGTTTACTGTTCATTCTAGCTGAATCAGTGAGCCCACTGAGAGACAAAAAATGATGAGGAACACTTGATGCTGACCTCTGACCACCACACACATGGGCACAGGAGGACACAAACATTAAAGGCACTTGATGCATCTCTGGGTCTTGGTCCCCTGATGAgcagctcctccagcctctatgaggccttgaccactttgtttttgagacattaacctggagttctgttgcagagagaccacaggcaagtgctcagctccctgctgtctggagccctggctggtgcacttgccaagacagcagtagcccccctggaccggaccaagatcatcttccaaggtaacagctgtgtgactgatcttcctcccacttctccgccaaccccaaatacagagtcccagattgtagcccagacaggcctcacatttcactttatagtctcggtcagccttgaatctacagcagtcttcctgcctcagcctagtaagtactgggtgctatgccacaggagactgccaggaattgccagtctgatacaagaacaaagagaatctttattataccaatctgactcggatcacaagccttctctgtcccgcaggttagggatGCGATGCTCTCACCCAGGGCCTGAGATATCTATAGGAGTGTGCACAGCTGGGATTCTTGAGCTTTGTACCGAGGTACATGGCTGGGAGGGGGAAGGAGTGCCGTCCTTCATCCTGGTAACAAGTGTTCAGACAGAGAGGTGGGGACAGAACTGCAGCTGGAAAGGGACCTTCCTGACCTGGGAAGAAGGAACTCCTTTTTGTTCGCAGTTTACAAACTGGTCTCAGCTGTCTGGAAACACTTAATTGCCTTTCCCATAGCCCAAGAGTGGGCTTTTGTTACTTCAAGAATATTGGCTCCTGCTTCCTTCAAGGGCAGGAGCCATCTGCACTGGAGAAATGCTGGTTGTTAATCAGGGGAGGGAGGGCAAGTTATGGCCACGGGGAGGCTAGGGTAGAATGCC
This mutation is a 448 bp deletion beginning at Chromosome 8 position 70,191,590 bp and ending after 70,192,037 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 48865 | TGC ATC TCT GGG TCT TGG TC | Common | A | |||
| 48866 | CTC TCT GCA ACA GAA CTC CAG | Wild type Reverse | A | |||
| 48867 | ACC TCG GTA CAA AGC TCA AG | Mutant Reverse | A | |||
| 48868 | Fluorophore-1 | CAG CTC CTC CAG CCT CTA TG | Quencher-1 | WT Probe | ||
| 48869 | Fluorophore-2 | ACC CAG GGC CTG AGA TAT CTA T | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 48865 | 0.40 uM |
| 48866 | 0.40 uM |
| 48867 | 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.