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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
>chr2:31674438+31674540 103bp TCCCTTCTGTTTGTTCAGCC CAATGTCTCCGACCTCTCCA
Mut= 103 bp
Wt= 104 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
CACCATTAGAACTCAGTGAGCTCACTGTAATTGAGGGAGCTCTGACCAGAAAAGAGACACAGTCCAATGCCTGGGCCTTAACAAAGGGCATTTCACCCTTGGAATCAGGGTGAGGGACCAGCAAAGTGTCTGAGCCAGGCAGGCAGTGTGGACATGTAACAGCTTGCAGTGAGCAGATGCTTTAAGGATAGCTAATGAGAGTCTGAAGTTCCCAGGTCTAAACAGTAATTATACCCATGTGCTAAAAAAGATGGTTATTGCCTCCCTTCTGTTTGTTCAGCCTCAGACACTGTGTGGCCAGCCCGCTggctattggtaagcttctgttttctcttgcagatacaatggagaggtcggagacattgtagtggggagaatcacagaggtaaagtcagtgttcataaagagaaggaggctggcggctcagggattggggccttcatctgtgaaagtatgaagaaggggccctttgtttctggggtggtgtgaaatgctatcccacatttatttctctcacaggttcaacagaagaggtggaaggtagagaccaattccaggctggactccgtcttgctcctctcatccatgaacctacctgggggagagctggtgagtgctcgcctgctctggaactgtccggtcggtcgggctgtggggctttgctccacctgagaagtctgcttcaCATGAGGTTGCTGTGGTTTTGTCACCAGATCTGCTACTTTGGTGAACACAGAGGCTTTTCTTCTAGCCCTGTGCTCTCAACATGGGCAGCAGCTTGTGTGTGGTAAGACCAGAAACACCCATGGCGTGGGCTCCCAGGAGGGAGCTGAGAGGAGGCTCTTGGGTCTTAGGATCTTAGTGCTGATTTCCTTTCTTGATGCCCAGCCTTGCACAGGGAAGGTGTCTCTTGAGGGGTTTGCCTTAGCTTGCTTGCTTTTTGGAGTGTGGGGTGGGTGGGGTTGAGACAAGGTTTCTCAGTGTAGCCCTGACCTGGAACT
This mutation is a 378 bp deletion beginning at Chromosome 2 position 31,674,483 bp and ending after 31,674,860 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 48640 | TCC CTT CTG TTT GTT CAG CC | Common | A | |||
| 48641 | CAA TGT CTC CGA CCT CTC CA | Wild type Reverse | A | |||
| 48642 | AAA AGC CTC TGT GTT CAC CA | Mutant Reverse | A | |||
| 48643 | Fluorophore-1 | AAG CTT CTG TTT TCT CTT GCA GAT AC | Quencher-1 | WT Probe | ||
| 48644 | Fluorophore-2 | CTC ATG AGG TTG CTG TGG TTT | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 48640 | 0.40 uM |
| 48641 | 0.40 uM |
| 48642 | 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.