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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
>chr10:108699986-108700111 126bp TGAGTGGAGTAGGCTTAAGATGG CCCATCACTTCCTCCTTCTCTG
Mut= 144 bp
Wt= 126 bp
Fam=Mut
Hex=Wt
SNP in Wt Reverse primer - rs254052209
Sequence
Wt Sequence (deletion in lower case):
TAACATGGTTAAGGGGAAAAAAAGAATCTCCCCATGTGGGCTCCACCCCCTGCACCCTAGGCTCCATCCACCCCCTGTGGCACCCAGCCACTCCCCTAGGCCTCAGGCTCCGCCCAGTGGAGACCCGCAAGGCCTCCTGGGAAGGCTACTTCTGGGCAGAGTGCAGTGGGCCCAAGTCTTGAGTGGAGTAGGCTTAAGATGGCGGTGCGGATGatgcagggagtgcaggcggtttaccttgaatcggacgctttcctggtttgtctcaatcacgctctgagcacagagaaggaggaagtgatgggtctgtgtatagggcagttgaatgatcacgggaggagtgactccaggcttgcatacgctggagctgagatgtgcacagttgcgaaaaagatggaggccaccagaattgttcacattcattctgttattatcttgcgacgttctgacaagacaaaggaccgggtagaaatatctccggagcaactgtctgcagcctcgatagaggcagaacggttagcagagcaaacaggtcgtcccatgagagttgtgggctggtaccattcccaccctcatataaccgtttggccttctcatgtcgacgtgcgtacacaggccatgtaccagatgatggatcagagctttgtaggactgatctttgcttgtttcatagaagacaaacccacaaagataggccgtgtactctatacttgcttccagtctgtacaagcatcgaagagttcagaatatgagagacttgaaatcccaatccatattgtacctcgtaccaccatcgggacagtatgcctcaggtcagccatagagctgccggggatcctatgtcaggaagaacaagaggcatataggaggattcacggccttacacatctggactcagtaactaagatccataatggctctgtattcaccaagcatctgtgcagtcagatgtccgcggtctgtgggcctctcctacagtggttggaggacagactagagcaaaaccagcagcgtttgcaggagttggaacaagaaaaggaagaccttatggaagagctgTCTTCCCTAGAATAAAGCAGGAAAACAAAAATGGGCCGATGTAAATACTTAGCATACGATTTATAAACTAATTCATTTCGACCTATTCTGACAAAGTACAAAGGTGATCCAAGGACGAAAGGGCACGGACTTGGGCTAACTGAACCTTTACAAGCCAACAAAATAAATTCTAACAGTACCTCAAAATGTTTCGTGGATTTAAAGCAAGATAGGAAA
This mutation is a 846 bp deletion beginning at Chromosome 10 position 108,699,232 bp and ending after 108,700,077 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 52815 | TGA GTG GAG TAG GCT TAA GAT GG | Common | A | |||
| 52816 | CCC ATC ACT TCC TCC TTC TCT G | Wild type Reverse | A | |||
| 52817 | GGA TCA CCT TTG TAC TTT GTC AGA | Mutant Reverse | A | |||
| 52818 | Fluorophore-1 | AGG GAG TGC AGG CGG TTT A | Quencher-1 | WT Probe | ||
| 52819 | Fluorophore-2 | TGC GGA TGT CTT CCC TAG AAT AAA G | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 52815 | 0.40 uM |
| 52816 | 0.40 uM |
| 52817 | 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.