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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
>chr9:58363982-58364075 94bp GAGCCCACCCCACATTAACT CACCTTTCACCCATCTCAGC
Mut= 92 bp
Wt= 94 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
AAATTCAGTCTTCAACATGAATCCTTCCAACTCAGGGGGTGCCAGAATTGTCCCCACTCTGCAGATGAGGAAACTGAGGCTTAGGGAAGGGAGCGCAACATTGCTCTGCAGGTATTCGGTTAAGTGGTGGCTCCAGAGTTCGGGGGCTACGTGGGGGGCGGGGACACTGAGCCCACCCCACATTAACTTGTCCCCACACTGTGCCAGCCAcatgttgggaacgcttgctcaaggccactcaggctgagatgggtgaaaggtggggaccaggtgggcaaggctaccccattccccttgaactcagcatcagctgctcccgaactatcccttcctggaaggggaaggtcaccttactcagccccaatgctgttctcaggaggaactataactccttgtgccagatgtatgagaaaatccagcccctgctggaaaacttacatgggaacttcacagagactcggaacaacatcggtgagtagagggagggagggagagaggaaggaagggctgggcaggggagagggagggagggctgggcaggggagagggagggagggctgggcaggggagagggagggagggctgggcaggggagagggagggagggctgggcaggggagagggagggagggaggaaggaagggctgggcaggggagagggagggagggagggctgggcaggggagagggcaggagccatttatgtcttttcctagtcatttccaggcaTAGGTGACTCCTGGGGCTTAGCAACTGCCTTGATTTTGGCCTGGGGTTTTCCTAGCCAAACCTGGGTCAATCTACCCGCCAGTGCTCATGAGTCCTACGTGTGCTTCCCTGCCACATGCTATCTCTGTTGCTTTGTCTTCTTCTCCCCACCCCTACGACTTTGAACTAAGTCAGATCTGAATTGGATGGGAGGAGCATTTGGGGGCGAGGAGAAGCCCAGAACAGCAGGGTGTGAT
This mutation is a 519 bp deletion beginning at Chromosome 9 position 58,363,515 bp and ending after 58,364,033 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 48206 | GAG CCC ACC CCA CAT TAA CT | Common | A | |||
| 48207 | CAC CTT TCA CCC ATC TCA GC | Wild type Reverse | A | |||
| 48208 | AAA ACC CCA GGC CAA AAT C | Mutant Reverse | A | |||
| 48209 | Fluorophore-1 | AAC GCT TGC TCA AGG CC | Quencher-1 | WT Probe | ||
| 48210 | Fluorophore-2 | CCA GCC ATA GGT GAC TCC TG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 48206 | 0.40 uM |
| 48207 | 0.40 uM |
| 48208 | 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.