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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
>chr6:114304271+114304370 100bp GGAACAGCCTACAGGAGA CCCTCACTGTGCCTAACA
Mutant= 115 bp
Wild Type = 100 bp
Sequence
Mutant Sequence:
CCCTGCCATGGAAACAGTGTGACAACCCGTGGAACACTGACCGCTGCTTCTCCAACTACAGCCTGGTCAATACCACCAACATGACCAGCGCCGTGGTGGAGTTCTGGGAGTGAGTGGGGGCTGGGGTGAGGCTGGGGAGGGTGTGCCAGCTGGGTGGTGGCAGGCAATGTGGTGTTTCCTTCAGCCATTCATTCCTGAATGTTTCTTGAATGCCTACTGTGTGCCGGTAGAGATCTGGGATTGCCAATAGACAGTGACAGCAGCAGGTGGCTCATATAGGCAGGGAGACGTGCAGGGTAGGGGAGGTGGCCTGGAACAGCCTACAGGAGAGTGTGGCTGCTCACCATCACATGTGGGGTTGGGTGGTCAGTGGTACTATAACTTCGTATAGCATACATTATACGAAGTTATATACTCTGTATCCTGTTTTCAGTTCATAGCCCATCTGTAGGGCGCAGTAGTCCAGGGTTTCCTTGATGATGTCATACTTATCCTGTCCCTTTTTTTTCCACAGCTCGCGGTTGAGGACAAACTCTTCGCGGTCTTTCCAGTGGGGATCGACGGTATC
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 69005 | Fluorophore-1 | TCA AGC TGA TCC GGA ACC CT | Quencher-1 | MUT Probe | ||
| 69360 | TCT TAT CAT GTC TGG ATC CC | Mutant Forward | A | |||
| 75711 | CCC TCA CTG TGC CTA ACA | Common | A | |||
| 76121 | GGA ACA GCC TAC AGG AGA | Wild type Forward | A | |||
| 76355 | Fluorophore-2 | AGT GGT ACT TAG TGG GGA GGA ACA | Quencher-2 | WT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 69360 | 0.40 uM |
| 75711 | 0.40 uM |
| 76121 | 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.