For in-depth product & services help, ask our
Technical Information Scientists
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
>chr7:28277753+28277831 79bp CGGTGCTTCTGAGGATTCA GTTCATGGGGCTCAGGTCC
Mut= 80 bp
Wt= 79 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
AATAATAATAATAATAAATTAAAGTATGTGATGCCGCCCCTGCTGGTTCAGAACTTGAGGCGGCCGTGGCGGTAATGGCACATGGCACCCCTAGAGGGCGATAGAGGTCCGGGCACCCGTCCTTGCGCAAGCACTTTTGTTTTCCAGGGCGCCGAGGCCCGCGCATGCGCACATCTTCACGCTGCCGCCGGAGCCGGTGCTTCTGAGGATTCAACGGTGGCAATGTCCGAGCTTCCcggagagagcaggatcccggacctgagccccatgaacggcgtcggcgacgtcctgcaggtgggactgggcggaggcagtccggtccccgaagcccaggaagacccgctggccacagcctcccagcccatggcgccggccccgggcctggctcccgggcagttccccggcgtggcgggcctgatggcgatcccgcacgtccacgcgccgctcctcctcctagagctgtacgagcagcggatgttccaacactatctgcacaccaaccccctgatccccaccaggctgctccacgaaatcgaggagcgccgccggctgttcgtggagggctgcagggccagggaggcggccttcgacgccaaccccccgcagatggactcgcacgcccgcgccttcatgctggcgctcacggcctccgacgcccgcggccccaaCGCCGACTGAGGGGTGGCCTTCACCTATAACCTCCGAGGAAGGAGGCCGGAGGACCGGACTTCAAGGTCATCCTCCGCCATGTAGCGAGTTGGAGGCCAGCCTGGGCTACAGGAGACTGTCTTTTAAAAATAAAAAATAAAAATAAAAGATTGGTGGCCAGGAAGTCAGGTGACTCCTAGAATCAAGGGCAAAGGCAGATGTCTGGGCAAATGTCATGAGGTGGGGAATCCATGCCAGAATCCA
This mutation is a 432 bp deletion beginning at Chromosome 7 position 28,277,795 bp and ending after 28,278,226 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 52615 | CGG TGC TTC TGA GGA TTC A | Common | A | |||
| 52616 | GTT CAT GGG GCT CAG GTC C | Wild type Reverse | A | |||
| 52617 | CTC GGA GGT TAT AGG TGA AGG | Mutant Reverse | A | |||
| 52618 | Fluorophore-1 | ACG GTG GCA ATG TCC GAG | Quencher-1 | WT Probe | ||
| 52619 | Fluorophore-2 | AGC TTC CCG CCG ACT GAG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 52615 | 0.40 uM |
| 52616 | 0.40 uM |
| 52617 | 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.