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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
>chr13:119702361+119702480 120bp CAGGTGGCTCTTCTGTTGC TGACTTTGGGATAGGCTAGGA
Mut= 118 bp
Wt= 120 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
GCTAGTATGTTATGACTCATATTTGAAAATCAGTGATACAGTCATATATAGCTGAGTGTCTAAATCCACATTTCAAACTATTAAGAAATTTAATTAGACAGGATTTGGGGTCACTGTTTAAGGACACAAGCAGGTGGCTCTTCTGTTGCCACCCGCCctgctctctttgcagtgtagacataaacttgttttgtttcttaaaattaataagtaaataaagatacgagcatcctagcctatcccaaagtcaaggcacagtttctggcttctgtggatgccatcatttcttggtcttctctttcagagggaaaggataaagattttaccctgaatgattttggcttcatgatctttcactcaccatattgtaaactggtgcagaaatctctagctcggatgttcctgaatgactttcttaatgatcaaaacagagataaaaacagtatttacagtggactggaagcctttgggtaagaacgctgttatgatgtgcttatttctattttataaatatttacatatatatagacctagagatacagtgagaaaacattctattctagttgagtggttgtggtccacgcctttacttccagcactcgggaggcagaggcaggcagatctctgaatttgaggacagccagggttacacagagaaactccgtctcaaaaaaaccaaaaaactttatttaatgaaaaccagttCTAGAGAGGTAGTAAGTAAATGGCTTATCTACACAAAATCATTAGTCATTTCTACTTAAGATTCTGGTGTCTGTTTTTAGCTTCAGCCTCATTGTTAGCGCACAATACTGTAATCACAGCCGCACCAGTGTACACCAAGCTG
This mutation is a 548 bp deletion beginning at Chromosome 13 position 119,702,388 bp and ending after 119,702,935 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 45646 | CAG GTG GCT CTT CTG TTG C | Common | A | |||
| 45647 | TGA CTT TGG GAT AGG CTA GGA | Wild type Reverse | A | |||
| 45648 | TGA GGC TGA AGC TAA AAA CAG | Mutant Reverse | A | |||
| 45649 | Fluorophore-1 | TGC TCT CTT TGC AGT GTA GAC ATA A | Quencher-1 | WT Probe | ||
| 45650 | Fluorophore-2 | CCC TAG AGA GGT AGT AAG TAA ATG GCT T | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 45646 | 0.40 uM |
| 45647 | 0.40 uM |
| 45648 | 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.