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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
>chr11:72266485-72266604 120bp GCAATCTTTTCTCCCTCCAG GAGTGGAAGCATCAGGATCG
Mut= 96 bp
Wt= 120 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence:
ccgccccaggaccgcagggaggcctggcaatggggggcggagttgtagccgcccccaatccggtgggaccgcgcgtcccccttttaagctcgtgaagcttctagctgcctcGCAATCTTTTCTCCCTCCAGTCAGTCTCCCTTTCACGCGATGGATTCGGCGAAGACTTGTGTGACCAAGTTCAAGTCCTTTGCGATTTTGCTCTTCACCCCGATCCTGATGCTTCCACTCGTCATTCTGATACCTGACAAGgtcagttgcgtgtctgggcagcctggcggaatccgggtgcccagtgcaataacgggcacggaacccgggaacgctgagctggaacgcatagattcc
Mutant Sequence:
TGGGACAGACACTCCCCACAGACCCCGAGGGGTGGCTCCGCCCCAGGACCGCAGGGAGGCCTGGCAATGGGGGGCGGAGTTGTAGCCGCCCCCAATCCGGTGGGACCGCGCGTCCCCCTTTTAAGCTCGTGAAGCTTCTAGCTGCCTCGCAATCTTTTCTCCCTCCAGTCAGTCTCCCTTTCACGCGATGGCCTCGGCGCTGAGCTATGTCTCCAAGTTCAAGTCCTTCGTGATCTTGTTCGTCACCCCGCTCCTGCTGCTGCCACTCGTCATTCTGATGCCCGCCAAGTTTGTCAGGTGTGCCTACGTCATCATCCTCATGGCCATTTACTGGTGCACAGAAGTCATCCCTCTGGCTGTCACCTCTCTCATGCCTGTCTTGCTTTTCCCACTCTTCCAGATTCTGGACTCCAGGCAGGTGTGTGTCCAGTACATGAAGGACACCAACATGCTGTTCCTGGGCGGCCTCATCGTGGCCGTGGCTGTGGAGCGCTGGAACCTGCACAAGAGGATCGCCCTGCGCACGCTCC
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 67647 | GCA ATC TTT TCT CCC TCC AG | Common | A | |||
| 67648 | GAG TGG AAG CAT CAG GAT CG | Wild type Reverse | A | |||
| 67649 | GAC GAA CAA GAT CAC GAA GGA | Mutant Reverse | A | |||
| 67650 | Fluorophore-1 | ATT CGG CGA AGA CTT GTG T | Quencher-1 | WT Probe | ||
| 67651 | Fluorophore-2 | CCT CGG CGC TGA GCT ATG TC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 67647 | 0.40 uM |
| 67648 | 0.40 uM |
| 67649 | 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.