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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
>chr5:125431742-125431836 95bp TGTCATGAGTAAGCATGGAGGA TCAACTCCTTTCAACACATCC
Mut= 95 bp
Wt= 95 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
GCCAGGTTCCCTCCCACCTGAGCTACATCCCCAGCCCCACCCCCAGCCCCTCAAGAATGTGATAGCTTGGTAATGCTAACAGTTGCAGCACACAGACCCTTGTGCAGCACTCAGAGATTGGTAGGTGACCTTGGCCACCTGCAGGAGGTGGTGACCTCATTTGATATTTGTCATGAGTAAGCATGGAGGACGCCCTGTGACTTTTTGCCCCCTAGTgagactctgttcgtcttttcaggcaaggatgtgttgaaaggagttgatgaaagcaatgttctggtgctgcctgggaagagaaagaagaagaccaaggcgccgcccccatccaagaaagagaggaagcccctgacaaagaaggagaggaaggtgctacagaaggtcttagaacagaaggagaagaagagccaggtacacctcactgtggctggtctgcaccagctctccagatCAGGGGGCTGGAGGGGAGTTTGTCCCTCCTTGTTATCTCTGCAGCAAGATTGTCTACTAGAACTTCCTGAAAGGAGGGAAGTGTTTTGTGTCTATCCAGAGACACAGTAGCCACTAGCTACAGGTGGCATCATTGGGAGCCTCTGGGTGTGCTGGCCTGATTTCAAAATTAGGTACATAAAGAGATGTGTACGTAAAGCATAAAGGTATGCGTCAAA
This mutation is a 232 bp deletion beginning at Chromosome 5 position 125,431,557 bp and ending after 125,431,788 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 43990 | TGT CAT GAG TAA GCA TGG AGG A | Common | A | |||
| 43991 | TCA ACT CCT TTC AAC ACA TCC | Wild type Reverse | A | |||
| 43992 | TTG CTG CAG AGA TAA CAA GGA G | Mutant Reverse | A | |||
| 43993 | Fluorophore-1 | CTC TGT TCG TCT TTT CAG GCA | Quencher-1 | WT Probe | ||
| 43994 | Fluorophore-2 | TAG TCA GGG GGC TGG AGG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 43990 | 0.40 uM |
| 43991 | 0.40 uM |
| 43992 | 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.