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
>chr18:61009177-61009277 101bp CTTTGAATTCCCAGCCTGATAC TTCCACCTACCATAATAGGCAAC
Mut = 108 bp
Wt = 101 bp
Fam = Mut
Hex = Wt
Sequence
Wt Sequence (deletions in lower case):
TTCTGATGCCTTCCTCAGGGAGCCCACATTCTCTGAAATCTTTTGCTTCCTGTGCCAAGGGTACCCTGGCTTCTTCCCTCTGTCCCCCTTGGAGCCTCTTGTCCTGAACACCTTCCAGACTGGCCACCTGTCCTCCGGTAAGCTCCACACTGTAGGCAAAGACTGAGCCATTAGCATTAGGAGTTCTGATGCCCCGCCTGGTTCTCCTGGCCAAGCAATCAAAACCCTCAGGTCGTTCATGACCCCTCCTGGAGCCTCTGCTTTCTTCACACTTGGAATAGAGTGTGGGTGAGTAGTTGGCTCAGGTGTAGAATGTTTACTGCTATAATCACTAACACGTGGTGACAACACACAGTGACTGCCACAAAGATCTTCACTGGCAGTGATATTAAAGTGGgtcatccttggcctatttctgctagccaagatggagacgggcagcttcctaaggcctgagccagctgccggcccctaaggatctgcaagctaggccctcttgagtccaggtgaaatcaccggatcatctgaacctgatgcgagatttcccactcaaccttgctcaacccacggtctcagtttggcctcaccctcctccctctctgctttttttcttctctctctctctcttactggcagcccatcaccccagacctgctgatgactcccagtgaccagggcgatgtggacctggatgtagacttcgcagcagacaggggcaactggacgggcaagctggacttcctgctgtcctgcatcggctactgcgtgggcttgggaaatgtctggcgctttccctaccgagcctacaccaatggaggcggtatgggcctgggaacctcttgggagtagctggggagacagaccaaggcttgggggctataggatggcctttggagtgaccttaggttagagggaggtggcttactctgcctgtcccttttcaccgcctaagcccctcctcctgctttgaattcccagcctgatacccaggtttcctccctaagacctaaagctttcctagaacctatagtggGCAGACATTGTTGCCTATTATGGTAGGTGGAAAGGGTAGTACCCACTGTCACTATTCATTGGCTTTGCAGTGTATCCTGTCCCCTCCCCCACCTCTCCCAGCGCCCTGCTCTACCCACAGTTCCCTTTCTAGAATCTTCCTTGTTCCCTCCTCTGCTGATTCTTTCCATTCAGACTGAAAAACGCAAGATCT
This mutation is a 636 bp deletion beginning at Chromosome 18 position 61,009,209 bp and ending after 61,009,844 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 48905 | TTC CAC CTA CCA TAA TAG GCA AC | Common | A | |||
| 48906 | CTT TGA ATT CCC AGC CTG ATA C | Wild type Forward | A | |||
| 48907 | GCT ATA ATC ACT AAC ACG TGG TGA C | Mutant Forward | A | |||
| 48908 | Fluorophore-1 | CAG GTT TCC TCC CTA AGA CCT AA | Quencher-1 | WT Probe | ||
| 48909 | Fluorophore-2 | CAG TGA TAT TAA AGT GGG CAG ACA | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 48905 | 0.40 uM |
| 48906 | 0.40 uM |
| 48907 | 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.