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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
>chr11:100632619-100632710 92bp AACCCCTCAGAGCAAAGGTC TGGTTGAAGAGCCAGAGTCA
Mut= 91 bp
Wt= 92 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletions in lower case):
TGAGTGAGGTGCTGTGACTTGCCTAGAGATGCAGAGCTTACATAAATGGCCAAGTTTGATTCTGAGGGCAGGCTGCCTGCATGGGAGCCCCTGTGTGAGAACAAGCTCTGTTGTAAACCTCAGGGCTCTATACATGTGGCAATGATGGTGACACTGACATCACCCCTTTGTGGCTGAGCCTTGTGTTCGTCCCTTTGACCACATATTCCTCTGGCCTATCCCCCTACTCAGCCTCCctcctagatgaggcatgagacccacgggaagaagatgacccctcccctccctcttccagaaccaggctgaggctcattacaagggccacagacacgccagaaaactcaaggctgtggaagctgccaagagcaagcagaggcctcgaaacccgaccacaaacgggacagtggtgtcttcagcctcacctccagccagtggaagccctggaacccctcagagcaaaggtcagtcttgatttcaagttcacacaacgaggCTGGCTTCTGACAGCGGCTTGAGTGACTCTGGCTCTTCAACCATCTTTCTTTCCTTTTTCTTTTGTTTGGTTTTTCAAGACAGGGTTTTTCTGTGTAGTCTCAGCTTTCCTGGAACTCACTCTGTAGACCAGGCTGGCCTTGAACTTATAGAGATCCATCCACCTGCCTCTGCCTCATGACAGTGCTGGGATTAAAGGCATGCACCACTACTACCTGGCTTTTT
This mutation is a 257 bp deletion beginning at Chromosome 11 position 100,632,662 bp and ending after 100,632,918 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 64400 | AAC CCC TCA GAG CAA AGG TC | Wild type Forward | A | |||
| 64401 | TGG TTG AAG AGC CAG AGT CA | Common | A | |||
| 64402 | GTC CCT TTG ACC ACA TAT TC | Mutant Forward | A | |||
| 64403 | Fluorophore-1 | CAA GTT CAC ACA ACG AGG CTG | Quencher-1 | WT Probe | ||
| 64404 | Fluorophore-2 | CTC AGC CTC CCT GGC TTC TG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 64400 | 0.40 uM |
| 64401 | 0.40 uM |
| 64402 | 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.