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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 transgene genotype is determined by comparing ΔCt values of each unknown sample against known homozygous and hemizygous controls, using appropriate endogenous references.
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
Mutant= 127 bp
Wild Type = 142 bp
>chr11:96892447+96892588 142bp CTTGCCCTTGAGGCCTTTCT CAGCCAAGGCTACACAGAGA
Sequence
Wt Sequence: ggagcagatggagtggaacgagtacggagttccagtccaccaacctggacccttgagtggagttagcttctgctaactcggtttacttatgtgtgacctgagaataagacctttctcagggccatttaaagtgGCTGACAAAGGGTCCGGGCAGTGTAAGTTCTGGGTCTTTTTTTTTTTTTCACTGAATTCTTCCTCTCTCTTCTTTACCTCCAGTGTACCTATATCGAGGTGGAACAGGTGGGGAAGACACACGCTGTGATTCTGAGCCGCCCCTCTTGGCTGTGGGGAGCTGAGATGGGGGCCAACGAGCTTGGTGTCTGCATTGGAAACGAAGCAGTGTGGACCAAAGAGCCTGTGGGGCAGGGGGAAGCCCTGCTGGGTATGGACCTGCTCAGGTAGAGAGCTGCTCTTCAGTGCCCCACTTCCTCCCACCTAGCCTGAGTCTATTGGGCTTGTCTCTGTTGGCCTTGCCCTTGAGGCCTTTCTAAGGCGGCTGCAGTCTTATATCTTCCTAGTCTGATAAAGGACCTCCCTTGgcctagagtgctttaatttttttttcagtactttttgttttgagacagggtttctctgtgtagccttggctgtccaggaatttgctctgtagaccaggatggccttgaattcacagagatcctcctgcctgtgtgccaccacacatagttcagtacaacttttgtttgtttgttttttggt
Mutant Sequence: GCTGACAAAGGGTCCGGGCAGTGTAAGTTCTGGGTCTTTTTTTTTTTTTCACTGAATTCTTCCTCTCTCTTCTTTACCTCCAGTGTACCTATATCGAGGTGGAACAGGTGGGGAAGACACACGCTGTGATTCTGAGCCGCCCCTCTTGGCTGTGGGGAGCTGAGATGGGGGCCAACGAGCTTGGTGTCTGCATTGGAAACGAAGCAGTGTGGACCAAAGAGCCTGTGGGGCAGGGGGAAGCCCTGCTGGGTATGGACCTGCTCAGGTAGAGAGCTGCTCTTCAGTGCCCCACTTCCTCCCACCTAGCCTGAGTCTATTGGGCTTGTCTCTGTTGGCCTTGCCCTTGAGGCCTTTCTAAGGCGGCTGCAGTCTTATATCTTCCTAGTCTGATAAAGGACCTCCCTTG
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 37064 | CTT GCC CTT GAG GCC TTT CT | Wild type Forward | A | |||
| 37065 | CAG CCA AGG CTA CAC AGA GA | Common | A | |||
| 37066 | CGG TTT ACT TAT GTG TGA CCT GA | Mutant Forward | A | |||
| 37067 | Fluorophore-1 | CTT CCT AGT CTG ATA AAG GAC CTC CC | Quencher-1 | WT Probe | ||
| 37069 | Fluorophore-2 | ACC TTT CTC AGG GCC ATT TAA AGT | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 37064 | 0.40 uM |
| 37065 | 0.40 uM |
| 37066 | 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.