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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
>chr1:171419641+171419748 108bp CCAGTTTGAGGGGATAGCAG CTTAAGCAGGCAGTGGAGGA
Mut = 99 bp
Wt = 108 bp
Fam = Mut
Hex = Wt
Sequence
Wt Sequence (deletions in lower case):
CCTGGGGCACTCAACATCCCTGGTATAAGATTTCCCTTGTTGGCAAAGGAACAGTCCATGATGGGCTTAACTAAAGGAGCCTCATTTAGTATGCTGTGGGGACGGTGCTGGTAGACATAGGGCCTTATATTTGCTCAAGCCAGCCCTCAGGTTCCCTAGAGAAAGGCTAAATGGAGACATGCTGGCAGCGGAACTAGCTTATCCAGTTTGAGGGGATAGCAGGTGGCCAGAGGGGCAGGTCCCCATctgaatgccgacttccctgaagctggatgctttaatccacctgatcctccactgcctgcttaagtctcagcaagtggcagtgtttgctggcctgactttgcacttgtgcgcctttgtctttgaggatggggtggggtgggggcagccactgtatgttattcggacagtctatgcaaatttgcctgctgtcaggattccgctctttcagcaccccctcggtgtacccctggctttcctttttgtcccctctcagtgtctgaattggaaatcgccttgaacatggacccagctgcttttcagactctgtactgtgctgagaagccaaagctagaagacaagaatcttattttcttctgtcagatgggcaggcggggtctccaggccacacaactggcacaaggtcttggctatacagggtatgacaaacagaaggcttgctagctgggtgctgcctggggactgtctcctggaccggtccttccctcaacatttctttttctccacagggctcggaactatgccggggcctataaagagtggctggagaaacagggctagataaaaggctcaccagtttgtggctgttctcagactaccttgctgggctaggcaaatccttgtagtgcttcacacatgaaaaactcaaataaaaagtacacttaatgaagtatgagttgtatttaatttgtcacatggacaaaaaaaaatggttctagtattaatctagtgctgtttatctcaggatctttgtccattcctctggtccctaccaccCAATTAAAGCAGTCCCCTGAATAACTGAACCTGACTTTGAACAGCAGTTTCCAGCCCAGAATGCACATCAAATAGTTACCTAATAGCCTTAAAAATAATCTTGGAGCTAGGTTGTGGGGCAGAGGACTGTGCTGCCAGACTGAAAACCTGGTGAGGTTGAGTGGAGTTCCTGCACTGGAACCCATGACCAGGACACGCCATTGAGAGGGCTATGACAATCAGGCATGTAGGGACAACACCTAAAGTCTTTCCGCA
This mutation is a 774 bp deletion beginning at Chromosome 1 position 171,419,685 bp and ending after 171,420,458 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 48939 | CCA GTT TGA GGG GAT AGC AG | Common | A | |||
| 48940 | CTT AAG CAG GCA GTG GAG GA | Wild type Reverse | A | |||
| 48941 | GCT GGA AAC TGC TGT TCA AAG T | Mutant Reverse | A | |||
| 48942 | Fluorophore-1 | CTT CCC TGA AGC TGG ATG C | Quencher-1 | WT Probe | ||
| 48943 | Fluorophore-2 | CCC CAT CAA TTA AAG CAG TCC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 48939 | 0.40 uM |
| 48940 | 0.40 uM |
| 48941 | 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.