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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
>chr5:31465595+31465754 160bp TGAGAAGAATCAAGCCTGCTG TGCCACGATAACCCATTACC
Mut= 160 bp
Wt= 160 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
AACCTAATCACTGTAATCCTTCATAGGCACGTCTAGGCTAATCTAATCTAGAAGGTTCCTCACAGGTATGCCCTCCCTTGATTCTCCAGCCTCTTGAATGTAAACTCTGAGTAGTCTGGGAAGGCCTGGGCTGCAGAGGGATCAGTTGTGTAGCCTGCTTGCTCTGACTGCCCTGGGTgtaagaggaattgtgtacactgtgttgaattgacttgtaattgttagtatgttttaacccatgtacttttttttgtatgtggtttctttttgttttatttatttatttatttatttatttatttatttatttattttgctttttgtaaacaaaacaaagaccagtcttcatagttttacacaagttattagcttggttgtatttttacaatatgagaaaaattagtaggaaatgtctccagcttctcctgtgtggtccatgactttgtgttgtatcagtaatagggcccagtgctccataaaggataacagtttccagtacaccatccctcatgaggactccctgagtggctcgtcatccgcctcctcttgtgaaccagtcagtgacttcacagcaaccctccgaaaatctacctactggatgaagatgagaagaatcaagcctgctgccacttcacaggttgaaggtatccatttcagattctcagctctgagacaggagagtgtatctagtgtgcttgatattttggtatggtatggttgtttatcagtagtgaggtgagggctgggtaatgggttatcgtggcaatgtatgccagagagtcaccatcagtggttgatttatagctgtctttgttgagtgctgcttctgtagacccatttaagcataaggaagatctaatggtttcTGAGGGAAGAGGGGACTCCATGGATTAAAAACAATTCACACCAGTGGTGGCACACGCCTTTAATCCCAGCACTTGGGAGGCAGAGGCAGGTGGATTTCTGAGTTTGATGCCAGCCTGGTCTACAAAGTGAGTTCCA
This mutation is a 688 bp deletion beginning at Chromosome 5 position 31,465,168 bp and ending after 31,465,855 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 42045 | TGA GAA GAA TCA AGC CTG CTG | Wild type Forward | A | |||
| 42046 | TGC CAC GAT AAC CCA TTA CC | Wild type Reverse | A | |||
| 42047 | CTG CAG AGG GAT CAG TTG TG | Mutant Forward | A | |||
| 42048 | TGG CAT CAA ACT CAG AAA TCC | Mutant Reverse | A | |||
| 42049 | Fluorophore-1 | ACT TCA CAG GTT GAA GGT ATC CA | Quencher-1 | WT Probe | ||
| 42050 | Fluorophore-2 | CCC TGG GTT GAG GGA AGA | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 42045 | 0.40 uM |
| 42046 | 0.40 uM |
| 42047 | 0.40 uM |
| 42048 | 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.