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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
>chr17:56010921+56011030 110bp CCTGGGCTCCAGTTTGTACT CCTCCAACAGCAGTTCTTCC
Mut= 119 bp
Wt= 110 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletions in lower case):
TGGGCTTTGATCCTGAAGGAGGTGGGGGTCATGAATACTATTGGCAGAGGGAACTGCTGCAGGGAGACTGTCAGGGGAGAGACAGGGCTCCGAGACAGTCAGTCTCACCCGAAGTAGTACAGCCAGGAGAAGACAGGCTGAATCCCAAGCGTGGGGAGAATCCGCACCTCGGACACCTCTGCTCACCGTCGCCctctggcggttggtccaggcaatggcacctcgggctcaagcctgggctccagtttgtactgagtgaagctcccagctgatgttgttgctacactccattcccaccagagtcccaccagtgaaggggaggaggaagaactgctgttggaggaggaggaagaagatgtgctggctggggtctcatcagaggacaaaggccacagaccccctgggaagggttccttggagccaggtgaggggccagggcagaggcactatgatgtgggaagatggctttgatgtttcttaggcctggggaatcctgaatcctgattcatattcatattctctctctctctctcattctctctctctctctctctctctctctctctctctctttctgtctgtCTCTGTCTCTGTCTCTCTCTCTCTGAGACAGGTTCTCATTGTAGCCCTGACTGGCTCAGAACTCACAGAGATGCATCCTCTGAGTGCTAGGGTTAAGGGCATGTATTATCTATTTATTGTGATGGGGCATGTAGATCAGAAGTTGGGTCCCTTCTTCCACCATGTGGGGTCTAGGAATCAAACTCAGGTCATCACACTTGGTTGCAAGATCTGCTGAGCCATCTCGCTGGCCCTG
This mutation is a 389 bp deletion beginning at Chromosome 17 position 56,010,881 bp and ending after 56,011,269 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 64346 | CCT GGG CTC CAG TTT GTA CT | Wild type Forward | A | |||
| 64347 | CCT CCA ACA GCA GTT CTT CC | Wild type Reverse | A | |||
| 64348 | AAG ACA GGC TGA ATC CCA AG | Mutant Forward | A | |||
| 64349 | GCC AGT CAG GGC TAC AAT GA | Mutant Reverse | A | |||
| 64350 | Fluorophore-1 | ACC AGA GTC CCA CCA GTG AAG | Quencher-1 | WT Probe | ||
| 64351 | Fluorophore-2 | AGA ATC CGC ACC TCG GAC AC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 64346 | 0.40 uM |
| 64347 | 0.40 uM |
| 64348 | 0.40 uM |
| 64349 | 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.