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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
>chr4:21893103+21893270 168bp AGGGTTGGAAAGCTTCAAGG GGAAACAAGGCACTCTCCAG
Mut= 170 bp
Wt= 168 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence: (deleted region in lower case)
GCTCATTGAGTGAGAGCATTTGCTGGTTTTGGAGAGGACTAGTTTAGTTCCCAAGACCCATGTCAGATGGCTTGAAAGTGCCTGTAGCTCCAGCTCCATGGGATCTAGCATTCTTTTCTGGCCTCCACAGGCACTTGCACACACTCACACACTCCACAGAGTGACAGATTTTACATATTCACTACTTAATTTTGATAATTTTAATTTTTAGATGGAAAAAAGTAAAACCTTTATTTTATTTTTTAATTAAATTCCAAATTAATGCctttgttatcccaaagaaactcctttgcttatcatttgcaatacaccatgagaatatcataaaaattcttcaactgtgttggtatcaggactcacatgctcctctctgcttttaacctttgttttcagcttactcagggacgcagctcactaggaatctacagattaagccgtgggaactcggtgaacacgggaccatgtgctttagatcctacaggatggctctctcctgtttgagcagagtaaaaacttacaggtgagtctgtcaattattctagtagagatttacatattttgtgtaacagcagtttagttttgattttgtttaataacctttgccagtatgttaaagcttatgtggtctaatttaagtcgcttttacttgtttaaataattttaaataattattttttaataatttttagggttggaaagcttcaaggtcacctaaaagttagaagggaaatatagtacattagccatgaatggtatttatttacgttcagagacaattattatttttaatgaaaattagacacatttctttcaggttcaccagcCTCCTTCCACCCTGGAGAGTGCCTTGTTTCCCTTAATAACCCTTATTACTAGTGTTCTATTGCTGTGAAGAGACACCATTACCATGGCAAGTCTTATAAAGGACACCAGTTAACTGGGGCTGGCTGACAGGTTCAGAGGTTTAGTTCATTGTCACCATGGTGGAAAGCATGGCAGCATGCAGGCAACATGGTACTGGGAAAGGAGCTGAGAGGTCTACATCTGGATAGG
This mutation is a 559 bp deletion beginning at Chromosome 4 positive strand position 21,892,681 bp and ending after 21,893,239 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 38424 | AGG GTT GGA AAG CTT CAA GG | Wild type Forward | A | |||
| 38425 | GGA AAC AAG GCA CTC TCC AG | Common | A | |||
| 38426 | ACA GGC ACT TGC ACA CAC TC | Mutant Forward | A | |||
| 38427 | Fluorophore-1 | AGA AGG GAA ATA TAG TAC ATT AGC CAT G | Quencher-1 | WT Probe | ||
| 38428 | Fluorophore-2 | CAC TCC ACA GAG TGA CAG ATT TTA C | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 38424 | 0.40 uM |
| 38425 | 0.40 uM |
| 38426 | 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.