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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
>chr3:127567556+127567655 100bp AGATGGAAAGGCATTTTTGC GAAACTTCTATGGCAGGTGATTTC
Mut= 122 bp
Wt= 100 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
TAGTATGCGAAAGCATATCCCTATTAAGATCAGTAAGAGAACTGGAGGGAGGGCTCAGCAGCTAAGAATTTCCTGCTCGTGCTGAGGAACCTGATTTCGGTAAAGCATCCCCTTCTGGTCTCTGTGGCACCCGACATGAATATAGTGCATATATGCATGTGTAGACAGATATGCGTGCCAAATAAAGAAATCTGTCTGTCTAGTGTATCTATTTGAGACAGGGCTTCTCCATGTAGCCTTGGCTGTCCTGAAACTTACTGTAAACTAGGTTGGCTTTGAATCTACAATCTATCTACCTCCACTTTCTGAGTACTGGGATTAAAGGCATGTACCCACATATGGTCATATTTTTTTTTTAAAAGTAATCAGTAATGTTTTCTAAATATATTGAATTTTTGCCATAAGAATGTACCAgcctatacagaatggtatactcatactctgtatagtatactatacggaatctttactcatactccagattaaatgattatcaagatattactttttaaagtgtattgcttcagttttagatggaaaggcatttttgcaaaataccgtgatgcatatgacaaatgaggctgtttgattctttcattgcagccaggaaatcacctgccatagaagtttcgcgtgctctgctagagtgtccttgtgagcatcgggcttctcagcaggtacgtatgtatctctctctaactactatgtttttcttgtgtagaaaatagtgacagattcatatgcatgtagcatatactttgattatactttctcactactctcccctgcccccattctggtctgtaTTCGTTTAAGCATCATTCGTGTCATACAGTTGATAAGAATTGTCTTGTTAGGGCTCCTGTTGCTATGATTGTTACTATGATGAAACACCATCACCAAAAGCAGCTTGGGGATCCGAGTGGTTATCTCAGCTTACAGTTCCAGGCAACAGTCCATTACTGGGGAAGTCAAGGCAGGAACTCAAGTCAGGCAATAGGGCAGGAATCTAGAGGCAGGAGATGGTGCAGAGACCATGGAGGGAGGAGAGCTGCTTACTGGCTTGCTTGATCTGCTTTCTTATAGAGCCCAGGACTATCAGACCAGGGACATGCCACCCACAATCCTCCCCCATCAATCACTAATTAAGAAATTGCCTTTCAGCCACATCTTTTTTTTCTTTCCTTTGACTTCCACATCATAGTCAATCATTGAAGGAAGTCAGGAACCTGGAGGCTGGAGCTGATA
This mutation is a 392 bp deletion beginning at Chromosome 3 position 127,567,439 bp and ending after 127,567,830 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 48243 | AGA TGG AAA GGC ATT TTT GC | Wild type Forward | A | |||
| 48244 | GAA ACT TCT ATG GCA GGT GAT TTC | Wild type Reverse | A | |||
| 48245 | GGC ATG TAC CCA CAT ATG GTC | Mutant Forward | A | |||
| 48246 | AAC TGT ATG ACA CGA ATG ATG C | Mutant Reverse | A | |||
| 48247 | Fluorophore-1 | ATA TGA CAA ATG AGG CTG TTT GAT T | Quencher-1 | WT Probe | ||
| 48248 | Fluorophore-2 | TTG CCA TAA GAA TGT ACC ATT CG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 48243 | 0.40 uM |
| 48244 | 0.40 uM |
| 48245 | 0.40 uM |
| 48246 | 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.