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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= 97 bp
Wild Type = 106 bp
>chr6:38041123-38041228 106bp CCATGGTCCTCAAGGGTTC ATGGTCTCCACCGCATCCT
Sequence
Wt Sequence (deleted region in lower case)
GACTATTTGCCTAACCTGCACAAAGCCCTACATTTGATTCTCAGTACCACATAAACTGGGTACAGAGGTACCTGTCTGTAATCCCGTGTGAGAGGTAGAAGCCTGAGAGTCAGGAGTTCAAGGTCATCCTCAGCTACGTAGGCGTTTGAAGTCAGTCTAGGCTACACAAGACGGAATAAGAAATAATAATGTTGCATATACCTGTAGAGGTGGGCCATGGTCCTCAAGGGTTCCCCTGCCCCCACACCAAGTGgttctcatactctgccttccttgtcagagaccaagacattcacggtggaggatgcggtggagaccatcggcttcgggcgcttccacattgcactcttcctaatcatgggcagcaccggggtgagcacacttggagaagctctgtggctgtgattctgtcctcactgtttagcaccaagttcctgctgacccccagcactggactgcccattcccgggagtaggaatcctgtggaaattatttcatcagcctttgcagtgggttggctgggaacgtgtctgttctggtaattcaagactccatcttagaggtttacactgagctcttgggaagaagggaaaggactgtggggttactccacgtggagtagctgcaagCTCTGAGCATGTGTGCATTGGTAACACCTGGAGCACCTGCTGTCGGGAAGACTCTAGATCCCCTAAGAGACTCAGTCATTTCCTTCATATCAGTGGTATTTGTTTCTCCAAAATCCTTGGTGTTGTGTGTTAGGCATGGTGGGTGCATCTGTAGTCACTGTATTTGGCAGATAGCTATAAGAGAATCAGGAGTTTAAGGTAATCTTCTGCTAGGTGTTGAGTTCGAGGCCAGCTAGGGATATGTGAGATCTAACTTTCCTCCAGATGTACACAGT
376 bp deletion beginning at Chromosome 6 negative strand position 38041189 bp and ending after 38040814 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 38035 | CCA TGG TCC TCA AGG GTT C | Common | A | |||
| 38036 | TCT AGA GTC TTC CCG ACA GCA | Mutant Reverse | A | |||
| 38037 | ATG GTC TCC ACC GCA TCC T | Wild type Reverse | A | |||
| 38038 | Fluorophore-1 | TGA GCA TGT GTG CAT TGG T | Quencher-1 | MUT Probe | ||
| 38039 | Fluorophore-2 | TCT GCC TTC CTT GTC AGA GAC | Quencher-2 | WT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 38035 | 0.40 uM |
| 38036 | 0.40 uM |
| 38037 | 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.