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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
Mutant= 120 bp
Wild Type = 111 bp
>chr13:104549546-104549656 111bp GGCGTGTGTAAAGCACTTGT GACTGATCTCTCATCTCCACCA
Sequence
Wt Sequence (deleted region in lower case)
CACAGAAGCCACTGTGCCTAATAAGGGAGGTTTACCAGGTGCTCTTCACGTGTGACTCAGACATGGGACAGTATTGAGTAAAATGGCCATGTCATCACCTTCCCCTTCATCCTGCTAGGATATGCAGGATCTTAGCACAGTGTACTTAAGCATAGACGTGGTCCATCTTAGTCCTGATTACCAGCACTGTCCCTGTCTTGTAAGGCGTGTGTAAAGCACTTGTAAACTAGTAGTGCTCTATttagttgtggaaggcataagatagacttcagtttttcttgctttggtttcttggtggagatgagagatcagtcatcactttctagggttacttttggcgattctgtatttagctatttttatttccttatgtgctgttgagatgtcctcagattgataaacatgaatatcatactaacttttgtttcgtgctttataactattgtgtgtgtttgtgatatactaatatttttaatttagaataaatcactccaacaatgcaatagtaaaacctcctgagatgacacctcaggctgcagctaaggagttagaagaagtaatgaagcgagtacgggaggctcagtcatttatctcagcagccattgaaccaggtaaggccacagtgTTGTTGTACAGGACGTAGTCTAAAGACCATAGACTCTTAGAACTGCAAGGAATTGTAGGATTTCACTGCTCAGTTCCCTCATTGTACAGATCAGAAGACTGAAACTCAAGACATAGTAAGTGGCTTACTCAGAGCAACTCGGCACAAACCAGCTTTGCCCTAACACTGGGCCATGCATTTTAGCCACAGTCAGTGAGCGCTCTAGGAAAACAAGTCAGTGCTGAGGCTGCTCTGCTCAACAGCAGGGTGGGGTGTTTCTGCGAGGCTAGAAAGGAAGGA
384 bp deletion beginning at Chromosome 13 negative strand position 103,759,538 bp and ending after 103,759,155 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 38341 | GGC GTG TGT AAA GCA CTT GT | Common | A | |||
| 38342 | ATG AGG GAA CTG AGC AGT GA | Mutant Reverse | A | |||
| 38343 | GAC TGA TCT CTC ATC TCC ACC A | Wild type Reverse | A | |||
| 38344 | Fluorophore-1 | CCA TAG ACT CTT AGA ACT GCA AGG A | Quencher-1 | MUT Probe | ||
| 38345 | Fluorophore-2 | TGG AAG GCA TAA GAT AGA CTT CAG T | Quencher-2 | WT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 38341 | 0.40 uM |
| 38342 | 0.40 uM |
| 38343 | 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.