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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
>chr2:19235367+19235516 150bp ACAGAGCAGTGACCACATCC GCTGCCTTTTTGCTTTCA
Mut= 147 bp
Wt= 150 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletions in lower case):
TTCAAGGCCAATCTGGACAACACTGTGGAATCTGAGCCCCCTTCCCTCCCACGATGTTAATGCATCAAATAAAAATAATTAAAGATGCAAAATATAAGAGGTATATGTGACCCATATCAGGAAAACAATCAATAATCAATTAGAGGAAATAAAATAGGAAGCTAAAAAAATTCAGTTACCAAAAAGAGAAGACAGAAAAAAGAGAGAGTAGAGACAGATAGCAAGATGGTCAATAGATATCAACCATATTAATAATCATACTAGATTATTGGTTTCCTTTTTAAACAGGAACTTTGCAGCATACTTACAGAGCAGTGACCACATCCTTTTGTAAGACTGCAGCGTCTTTAAATTTTAAGccactgttaattttgatgtgacagttacatgaacctaattacttttcctttttctccttcagtttgacccaatcacaattgaaagcaaaaaggcagccactgtggtgctgatgctcaagtctccagaggaagacatcttagccaaagcctgcgaagccatctataagtttgctctgaaaggtttgttcttttggtgttaaactttctatctcgtttctgtatagtactatatatatataataatatacataccaggtgctctgataagaataaatgccacagaagcaaaaagagcagttagataaacttgcattggatcatacccaaagtataagacaaatatccatgtgtacaatgttgatataagagttaaactgaaggcggccccaccccttcctccctgctcCCTTCCCCACTGGCCcctttcACTCAGGGTATTCTCCATTTCCACTTTGTTCTTCTACCCCCTCCTTTTCCCTCCCAGCCTCCTTTGCCCAGGCTCATTGGCCCTTCCTAATTTCCTCGGCTTTACCGCCCATTTTCATGCTAATTTTGTAATTTCATCTTCCTTTACAGCTGAATAAAATTCCATTCAAATAAAATTTAAATGATCAACAGGGACAGATAGTTGTATTCAATTAAAAACCAAAACCAAAACCAAACTGTATGCTGCCTTGAAGAAAGATCTTTAGAATATTAAGGCGCAAATAAAAGGTAAAAAGATATCTCATGAAAGGACTAATCAATATAAACATGGAGTGATTATATTAA
This mutation is a 406 bp deletion beginning at Chromosome 2 position 19,235,420 bp and ending after 19,235,825 bp (GRCm38/mm10). In addition, there is a 6 bp deletion (CCTTTC) 15 bp downstream the 406 bp deletion.
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 48856 | ACA GAG CAG TGA CCA CAT CC | Common | A | |||
| 48857 | GCT GCC TTT TTG CTT TCA | Wild type Reverse | A | |||
| 48858 | CAA TGA GCC TGG GCA AAG | Mutant Reverse | A | |||
| 48859 | Fluorophore-1 | CTC CTT CAG TTT GAC CCA ATC A | Quencher-1 | WT Probe | ||
| 48860 | Fluorophore-2 | ATT TTA AGC CTT CCC CAC TGG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 48856 | 0.40 uM |
| 48857 | 0.40 uM |
| 48858 | 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.