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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
>chr9:103360031-103360139 109bp CAGGACTGTATAATTGGACCA AAGCACATTGAACTTCCCAA
Mut= 103 bp
Wt= 109 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
GAACTGATTCCCAAAAGTTCGCCCCTAAAGAGTTTAGTCTCAGCAGAGGGAGGCAGAGACAGGCAGGTCTCTGAGATGTCGATCAGCTTTGCCTACGTATCTGATTCCATGCCAGTCAGGACTGTATAATTGGACCATATTTTCAAAAGAAATGTATTTTTTTGTAGAAGTTTCATATggtggtgcatacatctagttccagttcttgggaagttcaatgtgcttgtttttcaatagtgaaaaggaagatgatgataatgagaagagagaagatccaggagataattgggaagaaggtggaggtggcagtggagcagaaaaatcttcaggtccctggaataaaaccgctccggtacaagcgcctcctgctccagtaacaggtgatcttctgttttccctccTGAGGGATGGGCATAGTGGATCCAAATGGATGCTGCTTTAGTGGAAGGAATGTAGAAATTGAGGGACATACTTTTTTTGTCCTGTAGGCTGTCAAAGTTTTGCTTTTGTTTTTGGTGCTAGGGAATAAAGCCAAGTAGTTCACCTCCTGCTATCATCCTACCACTAAATCACATTCTTGGTGCTGCACTTAGGTCTTGAAGCTTACATTCTCAGCAGAAGGGAGTGAGTAAAATTTCTTTAGA
This mutation is a 223 bp deletion of Chr9:103,359,855-103,360,077 (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 72710 | CAG GAC TGT ATA ATT GGA CCA | Common | A | |||
| 72711 | AAG CAC ATT GAA CTT CCC AA | Wild type Reverse | A | |||
| 72712 | CTA AAG CAG CAT CCA TTT GG | Mutant Reverse | A | |||
| 72713 | Fluorophore-1 | TGG TGG TGC ATA CAT CTA GT | Quencher-1 | WT Probe | ||
| 72714 | Fluorophore-2 | TGA GGG ATG GGC ATA GTG GA | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 72710 | 0.40 uM |
| 72711 | 0.40 uM |
| 72712 | 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.