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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
>chr15:81895045+81895152 108bp TGCAAACAAGCAGAACTTGG GAAGTAGCTTTGGGACTCAGC
Mut= 96 bp
Wt= 108 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
TCTTCAGCTTGCTACATAGCCAAGGATAATTTTAATTCCTGATCCCTGAGCCCTGCCTCCCATAATGCTGACATTCACTTGTTCTTCAAGTAAACTGCAAACAAGCAGAACTTGGGATCCTGCACACAGATGCTTCTGTGCCTGCTCACTGCCACACAtgtgagagggaaaagctgtgccaggctgagtcccaaagctacttccttctttgtgcccgtaggttgaaccggcctcttactctctcagagaagattgtatatggacacctggatgacccagccaaccaggagatcgagcggggaaagacatacctgcgtttacggcccgaccgggtggccatgcaggatgccacagcccagatggctatgctacagttcattagcagtgggctgcctaaggtggctgtaccatcaaccatccactgtgatcatctgattgaggcccaggttgggggtgagaaagacctgcgcagggccaaggtgagcccaagagggctggtggctgggtgtggagtgggggaagggagacagagcccaggctgcagaaggctgaggcactactgctgagctgcctgtcAGTCTTTCCATGGCACTGATGGTCTGAGGATGGAATCCAGACCTCATATGTGACAAGCAAGTGTTCTACCACTGAGCCATTCCCCAGCCCTGAGGGTTAACATCTTTTTAAGACTTGCTTTTAATTCTTCCTAATACTTAAAATGAAAATCTGATGCAAA
This mutation is a 418 bp deletion beginning at Chromosome 15 position 81,895,108 bp and ending after 81,895,525 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 53935 | TGC AAA CAA GCA GAA CTT GG | Common | A | |||
| 53936 | GAA GTA GCT TTG GGA CTC AGC | Wild type Reverse | A | |||
| 53937 | CCA TCC TCA GAC CAT CAG TG | Mutant Reverse | A | |||
| 53938 | Fluorophore-1 | AGA GGG AAA AGC TGT GCC AG | Quencher-1 | WT Probe | ||
| 53939 | Fluorophore-2 | CTG CCA CAC AAG TCT TTC CAT G | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 53935 | 0.40 uM |
| 53936 | 0.40 uM |
| 53937 | 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.