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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
>chr10:81634808+81634954 147bp GTGGGGAAAGGACATCGAC CTTGTGGGCGATCAACGAG
Mut= 145 bp
Wt= 147 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
GTGCCCTATCCCAAAACCCGCAGCCAGAGGCAGGCGCCAGGCAAGTGCTCAGGGGTGGGTGGGGAGAGCCCGGAGCAACTGCCCCTGTTCCTTGGTGGGGAAAGGACATCGACCCACTCAGTCCCTCGcacctctgggtgtgccaccgcagggccaagactggggcaaaagtgaccagcgactgctccaggcagtggagaacaacgatgtggccagagtggcctcgttgatcgcccacaaggggctggtgcctacaaaactggaccccgagggcaagtctgcgtgagtgtccaagccctggggctggggtggctttggacattcttctggggttgacccctgactcgtgcctctaggttccacttagcagccatgaggggtgcggcggggtgtctggaagtgatgctggctcaaggtgccgatgtcatgagcacggatggagcaggtacttatggaaggaccgtcccTGGGTGGATGGCAGGGCTGCATCAGCTGTGCAGAGCAGGGACCACAGGTCCAACTGTGGAAGCTCACTGTCCACAGTGGGAGCTCACTGTCCACAGTGGGGCTGTAGCTCAGTAGGTAGGA
This mutation is a 339 bp deletion beginning at Chromosome 10 position 81,634,842 bp and ending after 81,635,180 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 45073 | GTG GGG AAA GGA CAT CGA C | Common | A | |||
| 45074 | CTT GTG GGC GAT CAA CGA G | Wild type Reverse | A | |||
| 45075 | TGA GCT ACA GCC CCA CTG T | Mutant Reverse | A | |||
| 45076 | Fluorophore-1 | CAG GCA GTG GAG AAC AAC G | Quencher-1 | WT Probe | ||
| 45077 | Fluorophore-2 | CTG TGC AGA GCA GGG ACC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 45073 | 0.40 uM |
| 45074 | 0.40 uM |
| 45075 | 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.