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
Mut= 158 bp
Wt= 166 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
CAGGCAGAGATAGCTTGTGCAAAGCCCGGGGGCAGGAGCCTTCCTGTATCCGTCTGCGTTCCTGGTATGGAGGCTGTCGAAAGATTCCTTTGTTACTCTGGCTGTAGAAGAGAGACTGGTGGGAGAGAGTCTCTGCAAAGCAACTCAGTGGTGGGCACAGACAGggcgtgtgcctggggttaatgacataactattttggaaaatgagctttctatccaggaaaggggctccccatatgttagtttgcttgcttgtttctgaaggaaagagcccggggcacaatcagattgtgtgccaatcgttttgacatgcctcttgctcagaagggatgcctggccttgcttctgtcccaggaaggcatgctgacagatgcggagagagtaattctcccaccccatctctttcccctctccctcatcctctccctctccctccctcacaggtgctttcagaacttagagtggttattcatatatcaaaacctgctcaaagtaagttaccctatacttggaatgtcagaggactgcttatacctcaacatttatgcaccatgccatgccaataacggctccagtctcccggtaaggctgcctgctgccctggggttcattagcaggtatgggtaacagctgggcatggggcatctggagttagacctagcctcccattgggtaagaggaaaagtccatcacacaagtaacttgtttgttggttggttttttttcttttctttccctcgagacagggtttctctgtatagctctggctgtcctggaactcactttgtagagcagGCTGGCCTCGAACTCAGAAATCTGCCTGCCTCTGCCTCCCAAGTGCTGGGATTAAAGGTGTGCACCACCATTGCCAGGCACACAAGTAACTTTTAAAAACATATTATAATGGAAGCTGTCCTCTGTCATTGTCTCTGTTGAATATTTCG
This mutation is a 631 bp deletion beginning at Chromosome 8 position 93,530,546 bp and ending after 93,531,176 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 45562 | AAC TCA GTG GTG GGC ACA GA | Common | A | |||
| 45563 | CAA AAC GAT TGG CAC ACA | Wild type Reverse | A | |||
| 45564 | GAG ACA ATG ACA GAG GAC AGC | Mutant Reverse | A | |||
| 45565 | Fluorophore-1 | AGG AAA GAG CCC GGG G | Quencher-1 | WT Probe | ||
| 45566 | Fluorophore-2 | CAC CAT TGC CAG GCA CA | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 45562 | 0.40 uM |
| 45563 | 0.40 uM |
| 45564 | 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.