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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
>chr1:170302458-170302615 158bp TTCAAGCCTGGGCAGTGT TGTCCACGTTCCTTCCTCT
Mut = 165 bp
Wt = 158 bp
Fam = Mut
Hex = Wt
Sequence
Wt Sequence (deletions in lower case):
AAAAACAAAACAAAACAAAACCCTGGAAAGTTTAAAAACATCTTTACTGGAAGCTTCCAGaaggtttggggtatattgcaaaaccatcaaagctgagtgacaaatccagggtcctgttcccctccacttacatgtctctctctctctctctctctctctctctctctctctctctctctctctctctctctctctctctttcttttctttgtccatagttgaccacagtatgtttgagaatttgaacacgaccctcactccaaagctccagtccagccattcctttccccacctgtccagacctggggccccaggctctatcgcccctggttctggagagccaggaggaccaggactgagggtgggcagcagccagcaccttaggaatctgggcaaagctgtgggggccaaggtcaatgacctcctgcggagaaaggagtcttcaagcctgggcagtgtgggtgtattggagattaataagacggctggagcccagctgcctggtggggaagacgcagcctgtggaccctggctggaggatgaaaggtgagtcctctcaaccccccattatcccagTTGTAGAGGAAGGAACGTGGACAAAACCACAGAGCATACAGGAAAGATCAGACCCA
This mutation is a 516 bp deletion beginning at Chromosome 1 position 170,302,481 bp and ending after 170,302,996 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 53140 | TGT CCA CGT TCC TTC CTC T | Common | A | |||
| 53141 | TTC AAG CCT GGG CAG TGT | Wild type Forward | A | |||
| 53142 | GGG GAA CAA CTG AAA TGT TTG | Mutant Forward | A | |||
| 53143 | Fluorophore-1 | CTG GAG GAT GAA AGG TGA GTC C | Quencher-1 | WT Probe | ||
| 53144 | Fluorophore-2 | CAT CTT TAC TGG AAG CTT CCA GTT G | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 53140 | 0.40 uM |
| 53141 | 0.40 uM |
| 53142 | 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.