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
>chr11:62779690-62779821 132bp TTCGTCAGCTCTTATTCAACACC GGCTGCTCTTCAGTGTGAGT
Mut= 140 bp
Wt= 132 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
TGCGTTATATTTCTGATCCCCTTTCTCCCCTCCGATTCTCCTGGATAAAGCTATTCGTCTTCATATTCATCCCCCTCGATTTCCTTCTTATTTTACAAACCATCCAGTAAGAGCGTCAGATCTCCTAACTCCACAAAGGCATGCTTCCATCTTGATTCATCTTCACCTGTTATAGGACTGTGTTACTGTTGAGCACAGAAAACAAAGAACATGTGCATTTGCAATGTCTTTTCAGACATGGAAACcagacctgagagcaaggagtcaacttcagtgctagacttttccaaagaagagtcaaacagactgacagaaaatactgcttctgactccgacattgaaactgctcttgaatgtgagcgttggctagaacatcgtcaaagaaatcgggagaggtacccaagacaaatgtctgctcacaggaactccctccctgcgcaagaagatggtgaacgtgatgtatattggaaaagcttcagtcagaaacctctccttacaactgatgacagagaccccaaaggatcctgggacgttcacacgcttgggaaaagactgaagcagaaatcaaagttaatgacaaagcagagaccttataaagagaaaaaacctcataagtgtgatgagtgtggcgaactcttcacttaccgttcggtacttatccgacaccagagagttcacactggagagaaaccttatacctgcagtgagtgcgggaagtccttcagccacagagccaatctgactaagcaccagagaacccacactaggattctcttcgagtgtggcgagtgcaagaaaaccttcatagaaagttcatcccttgaggtacatcagagaatccacatcggggagagaccttacgaatgcgccgagtgtgggaaaggcttcaaccgaagtacacaccttgcccagcatcagctgattcatactggggtgaagccttatgaatgtaacgaatgtgacaaagctttcattcactcatcagctctcattaagcatcaaagaactcatactggagagaagccctataaatgtcaagactgtgggaaagccttcagccactgctcatctcttacgaagcaccagagggttcacacgggggaaaggccatatgagtgcagtgagtgtgggaagactttcagtcagagcacgcaccttgttcagcaccagagaatccacactggagaaaagccctacgaatgtaacgaatgtgggaaaacattcagccggagctcaaattttgcaaagcatcaaagaattcacatgggaaagaaaccctataaatgtggagaatgtggcaaagccttcgttcattcgtcagctcttattcaacaccagagaactcacactggggagaagcctttcaggtgtaccgagtgtgggaaaagcttcaagagcagcccctccctcatcCGACATCAAAGAACTCACACTGAAGAGCAGCCCTGAGAGGTGCTGAGTGTTAGGTGAAGGTTGGCTTTATTGCTGCGTTTCATATTTGATTGTTGAAATAGAGAGACCATAATATGACCCTGGGGTGTGTCTGTGTGTGTATGATTTCACTTGCCCTGTGTATAACTCTGAACAGCAGAAAGGAGATCCCTTCTACCCATTGGCTTGATAATAGTGAAGCCCTCT
This mutation is a 1168 bp deletion beginning at Chromosome 11 position 62,779,722 bp and ending after 62,780,889 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 52659 | TTC GTC AGC TCT TAT TCA ACA CC | Wild type Forward | A | |||
| 52660 | GGC TGC TCT TCA GTG TGA GT | Common | A | |||
| 52661 | GGC ATG CTT CCA TCT TGA TTC | Mutant Forward | A | |||
| 52662 | Fluorophore-1 | AGC CTT TCA GGT GTA CCG AGT GT | Quencher-1 | WT Probe | ||
| 52663 | Fluorophore-2 | CTT TTC AGA CAT GGA AAC CGA CAT C | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 52659 | 0.40 uM |
| 52660 | 0.40 uM |
| 52661 | 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.