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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
>chr15:98554425-98554525 101bp ATGGGAGAAAATCTTGTGAAGG AAGTGACCAGGGTTAATGATCTG
Mut = 99 bp
Wt = 101 bp
Fam = Mut
Hex = Wt
Sequence
Wt Sequence (deletions in lower case):
CTACATGAGTGTAGGGCACCACCTGCATACGTGCCTGGGAAGGCCAGGGAGTGAGTGTTGGATTCCCAGGAACTGGAGTTACAGATGGCTGTGAGCTTTCTGTGAGTTCTTGAAAGCAAACCTAGGTCTTCTGCAACAGCAGCAAGTGTTCTTAACTGCAGAGCCATTGCTCAGGGGCCTCTATCCAGACTTTTGATTGTTTTGTTTTTAAAGTTATTTACATCCTTTTGctggatgtaacttctgggttttttttccccttcttaggcttacctacaacaagttcaagatctggtgattttagagagtataattttgcagaccttaggtaagttaattttcctttttaaagcaggaaaaaaatatgtatgggatataatgatttcatgttaaagtttgttagatgggagaaaatcttgtgaaggctttgggaagtgtctgttcctgggtaaaaatgtctgTGGGGCTCATTAAATGGAGACAGATCATTAACCCTGGTCACTTTTCACAGTGTTAGGTCCTTGGTGACCCTTGAAACAGCTATTAGTCAAGATTTAAAAAAATTTTCTCATTGTAAGGTTTATGATTTTTTTAAAAAAAGTTTTATTATATATAAGTACACTGTAGCTGTCTTCAGACATCCCAGAAGAGGGTGTCAGATCTCATTACAGATGTTTGTGAGCCACCATGTGGTTGCTGGGAATTGAACTCAGTACCTCTGGAAGAGCAGTCGGTGCTCCTTAATTTTTATTTATTTATTTATTTT
This mutation is a 231 bp deletion beginning at Chromosome 15 position 98,554,468 bp and ending after 98,554,698 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 48929 | AAG TGA CCA GGG TTA ATG ATC TG | Common | A | |||
| 48930 | ATG GGA GAA AAT CTT GTG AAG G | Wild type Forward | A | Wt F | ||
| 48931 | GGG CCT CTA TCC AGA CTT TTG | Mutant Reverse | A | Mut R | ||
| 48932 | Fluorophore-1 | AGT GTC TGT TCC TGG GTA AAA ATG | Quencher-1 | WT Probe | ||
| 48933 | Fluorophore-2 | ATC CTT TTG TGG GGC TCA TT | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 48929 | 0.40 uM |
| 48930 | 0.40 uM |
| 48931 | 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.