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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
>chr15:4904759+4904890 132bp AGTATTCAGCAGGCATAGATGG AGGTGAAGAGCAGTAGGGAGA
Mut= 133 bp
Wt= 132 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
TAAAAAACGATGTCCACCATTTGTTCCTATAGTATTTTATTGAACAGATGTCATTGAGTACCTGTTTATTTGCTATACCTCTGGGACTTGGGAAAAGCTAGATGGAATATATGGATATTAGTGAAGTAACAATCCAAAATTGCTTATTAGTATTCAGCAGGCATAGATGGCTACCTAGCTAGTGGTTTCCTTCAgacccacggcaccttgattaatgatatttaggctttcagctgaaggaagaagagcaagaacataatctccctactgctcttcacctatctaagccagtaagtcctggcatgctttttatggatgacggaaagagtcaggaccacccacatgaaaagtgaatggtgccttttcctcttgtgcccttcactgcagtgtctcagagcatacccttcatgatgatgaccctgctcaccatgcagaccatgctcaggctggtagaggatgaaaatatgaggcaaaccttctgtataggtgggtattcctatgatttgctttctgctgaaaaataaacatgcttccaggacatttctcacatggtgcagatagatcacacagagtttccaaaagagtttatatcaaatcttcgctgccatggatgtgtatgagtaccacctataacttagtctagttgacatgtgtgtggcattggccgtgttaaatatgttccattctagacagactacctcctagtaagagtatataataacaatcttaatttatactctattcactactaattgaaattggctaaataattttcatgttttcatggttatttgatgaagttttaagatgatttagaaacttttgctaattgtctgtgctttcttttagaaaaatttgatgtttctttccatcttcacctttgtttcagtctgtggttgtattttttttatttttaatgcagcccttgaaaatttcagcaagtccatttacaagtatgtcaaccactggaaggattttccatatcccaaattagatgctaaccggctctctgacaagatcttcatgctgttccggtatataatggagaaatgggccccgcaggcctcacccatgcatgtgagtgaggtttcagataggctcaagttgtaaatccccatactggtcaaaccagttttagagtttggttctatcttatatatgtgcatcttacacgcatgcgcgcgcacacacacacatatgttctaaacatatatatatatacacacacacacacacacactcatatgtgtcctaaaatttagtgaacttctttaggttcttgccaatactttaaGCTATTAAAGCAGTTTTTAAAAGAATATTTCCCAGTTAAATCATCAAATATGGAACTTCTTATGGGTATCATTAGTTATGTTTTGGGAGGTATATTAAGTCTACCAATTTTTCTATCTATTTCAAGTCACTGTGTATCCATTGTCCCACTTGAAGTTTTTGAAGAACAACAATATA
This mutation is a 1100 bp deletion beginning at Chromosome 15 position 4,904,805 bp and ending after 4,905,904 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 44820 | AGT ATT CAG CAG GCA TAG ATG G | Common | A | |||
| 44821 | AGG TGA AGA GCA GTA GGG AGA | Wild type Reverse | A | |||
| 44822 | CCC AAA ACA TAA CTA ATG ATA CCC | Mutant Reverse | A | |||
| 44823 | Fluorophore-1 | CCC ACG GCA CCT TGA TTA | Quencher-1 | WT Probe | ||
| 44824 | Fluorophore-2 | TTC CTT CAG CTA TTA AAG CAG TTT TTA | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 44820 | 0.40 uM |
| 44821 | 0.40 uM |
| 44822 | 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.