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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= 105 bp
Wild Type = 136 bp
>chr2:121976707+121976842 136bp GAAATCCAAATGCTGAAGAACG TGCAGGCGTATGTATCAGTCTC
Sequence
Wt Sequence: AAACCCCTCAAATTCAAGTATACTCACGCCACCCACCGGAGAATGGGAAGCCGAACATACTGAACTGCTACGTAACACAGTTCCACCCGCCTCACATTGAAATCCAAATGCTGAAGAACGGGAAAAAAATTCCTAAAGTAGAGATGTCAGATATGTCCTTCAGCAAGGACTGGTCTTTCTATATCCTGGCTCACActGAATTCACCCCCACTGAGACTGATACATACGCCTGCAGAGTTAAGCATGCCAGTATGGCCGAGCCCAAGACCGTCTACTGGGgtaagcctcaagttcttccttactttctggacgctccatctgtgtggacttaaaactgctttgctattttaaaaacctgcatactgagattgttagagaataccc
Mutant Sequence:CCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGGGGATCggcaataaaaagacagaataaaacgcacgggtgttgggtcgtttgttcggatccatcgaTGAATTCACCCCCactgagactgatacatacgcctgcagagttaagcatgccagtatggccgagcccaag
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 35415 | TGC AGG CGT ATG TAT CAG TCT C | Common | A | |||
| 35416 | GAA ATC CAA ATG CTG AAG AAC G | Wild type Forward | A | |||
| 35417 | Fluorophore-1 | TCG GAT CCA TCG ATG AAT TCA C | Quencher-1 | MUT Probe | ||
| 35418 | Fluorophore-2 | CAG ATA TGT CCT TCA GCA AGG ACT G | Quencher-2 | WT Probe | ||
| oIMR8394 | AGG GGA TCG GCA ATA AAA AG | Mutant Forward | A |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 35415 | 0.40 uM |
| 35416 | 0.40 uM |
| oIMR8394 | 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.