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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
>chr5:135967847-135967972 126bp AGCGTTTGCGACGATGACT CAGGAAAATGGGCCCTCTG
Mut= 117 bp
Wt= 126 bp
Fam=Mut
Hex=Wt
Sequence
MUT Sequence (insertions with carrots ^g^ (T insertion):
tcccgattccccactgcaccagcttgtctccatttttttttttttttttttttttacagAACTGAGGCTGGTAGGGGGCCCGAGCCGCTGCCGAGGGCGCCTCGAGGTCATGCATAGCGGCTCCTGGGGCAGCGTTTGCGACGATGACTGGGACGTTGTAGATGC^T^TCCATCTGGGCTGGGGACAGCACAACTGCGGCCACCACGAAGACGCCGGAGCGCTTTGTGCAGgtgaagctgtggcaagagaggaggtggagccggagtggtgggcggggcccgacccataggaggtgcgggcagggtttatatggaggaaa
This mutation is a 4952 bp deletion beginning at Chromosome 5 position 135,962,986 bp and ending after 135,967,937 bp (GRCm38/mm10). In addition, there is a single bp (T) insertion at the deletion site.
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 47167 | AGC GTT TGC GAC GAT GAC T | Common | A | |||
| 47168 | CAG GAA AAT GGG CCC TCT G | Wild type Reverse | A | |||
| 47169 | CTC TTG CCA CAG CTT CAC CT | Mutant Reverse | A | |||
| 47170 | Fluorophore-1 | AAC GTG GTG TGT CGT CAG C | Quencher-1 | WT Probe | ||
| 47171 | Fluorophore-2 | ACA ACT GCG GCC ACC A | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 47167 | 0.40 uM |
| 47168 | 0.40 uM |
| 47169 | 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.