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
>chr10:42924204+42924348 145bp CTAGGTGGCAGTGATCCTAGA AGATGTCAGCCTGCCTTTGC
Mut= 135 bp
Wt= 145 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
GTCTTCACCAGTTCCATTAGTGTAAACACCATCTTCTCACGGTGACGTTTCAGGAAGACCCTCACAGTGGTGGCTACTCCTTCCGATCCCCATCTGCATTGTCTGATGGTTTGCTACAGGAAGTCACTGCTGAACAGCTAGTCTGAAACCTGGAGAGAGCTCATGCTGTGAGCTCATCAGAGAATGGGAACAGCACCATGGCCTATCTCCTGGTGGATACAGGGAGAGCCTACAGCTGGGATTCTACCTAGCACAGAGCTGTGGGCAGAGCACTGCCCTGCAGTCTGTGTTCTAATCTTGGGAGCTTgtgtgtcttaactgttcatgcctctctgtgttcctacacacgttgcctggtgctgtcatttcccgggcagtgtcaatgtaagggtacagagctctcctctgaggataatccaaatacaaattttgaacatgaacttgaatctgaagctctgttcccaaagctgaggagtaggaaggtgcaggagtgtgaaggtgtagagagagttagcaggcagccaggatggtcagggtggtatagagagcatttttagccatttgctgattgaactgactctcctgcagatcaagtctccagtgctcatgactcccttggccctgtcacctccacggagtaccccggaacccgatctcagctccatccctcaggatgcagccaccattcccagcctggtggtcccagaggctctcacaggtacgtctgccaaaggacttctaggtggcagtgatcctagagtaacccggcgtcctttcatctaatggttactttatgaatctaagagtttctctgaggtacttatttcactggGTGTGATGACACATGCCTTTAAACCCAGGAGGCAAAGGCAGGCTGACATCTGAGTTTAGGGCCAGCCTGGTCTACAGAATGAGTTCCAGGATAGTCAGGGCTACAGAGAGAAACCCTGTTTCTGAAAAGAAAATATAAAGAGAGAAAGGAAGAAAAGAAAAGAAAAGAAAAGAAAAGAGAAGAAAAGGAAAGGAAAGGAAAGGAAAGGAAAGGAAAGGAAAGGAAAGAAAGGAAAAGGAAAGAAAGGAAAAGGAAAAGGGAAAAGAAAAAGAAAAAGAAGAACAAAAATTTCCTGAGATAGCTCAAAGAAAGAT
This mutation is a 525 bp deletion beginning at Chromosome 10 position 42,923,773 bp and ending after 42,924,297 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 48694 | CTA GGT GGC AGT GAT CCT AGA | Wild type Forward | A | |||
| 48695 | AGA TGT CAG CCT GCC TTT GC | Common | A | |||
| 48696 | GAG AGC CTA CAG CTG GGA TTC | Mutant Forward | A | |||
| 48697 | Fluorophore-1 | CCG GCG TCC TTT CAT CTA A | Quencher-1 | WT Probe | ||
| 48698 | Fluorophore-2 | CTT GGG AGC TTG TGT GAT GAC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 48694 | 0.40 uM |
| 48695 | 0.40 uM |
| 48696 | 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.