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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
Mutant= 83 bp
Wild Type = 85 bp
>chr9:42449749-42449833 85bp TGAGACTCTGGCCATCTCTG TCACAGCAGTAAGCACCAATG
Sequence
Wt Sequence (deletions in lower case; bp changes in brackets with wt first and insertions with carrots ^g^ (g insertion):GATACAAGTTAGCTGGGACTGAGGTGTAGGCAAGAAGATCGTGACGCGTTGCTGGTGGCTGGCGACAGGCAGCGGGGAGCAGAGCCCAGGAGACTGGAGGGAGGCCAGCACCTCTGAGCTCTTCCTGGCCCAGATTTAACTCTCTTTAGGATTCAAGGTCCCAAGACCCTCTTGGAATGCAGGAGATAGGGTTTGTTTCCTTATGATGCCTGGGGCGAATGTACACAGAC[^g^]aggagcgaggctgagtggagcaagtgttttgtgtttccaataattcagatcgcctcaacctcccgagcgtgctcgtgttgaacagctgcggcattacctgcgcgggagatgaaagagaaatcgcagccttctgtgctcacgtgtcagaactcgatctttctgacaacaagctccaagactggcatgaggtaaagcctatgtaccccacctctgaggggggaaaggcgatgcttgttgcttgcttatgtaacttctgttctgctgactgtggtcctacttgagactctggccatctctgtgttagggtcaacagcaagctgccagctacctctggtgAATTCTCATTGGTGCTTACTGCTGTGACTCTGCTTTTATTGGTGTGCGTAGCGTGGCGACTTTCATTTCAGTCATCAGAAGGTGGATGCGTCTTCAGAAGCTCCGATTGCTCACGTTTCTGTTTCTCAGTCCCCTGTCCTCTGCTTCCACTTCTGTATCTCTGCAGACT
Mutation details
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 44567 | TGA GAC TCT GGC CAT CTC TG | Wild type Forward | A | |||
| 44568 | TCA CAG CAG TAA GCA CCA ATG | Common | A | |||
| 44569 | GAA TGC AGG AGA TAG GGT TTG | Mutant Forward | A | |||
| 44570 | Fluorophore-1 | CCT GGG GCG AAT GTA CAC AGA | Quencher-1 | MUT Probe | ||
| 44571 | Fluorophore-2 | AGG GTC AAC AGC AAG CTG CC | Quencher-2 | WT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 44567 | 0.40 uM |
| 44568 | 0.40 uM |
| 44569 | 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.