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
Mutant= 94 bp
Wild Type = 124 bp
chr7:30161703-30161826 124bp GGTAAAAAGCCTTATCAGTGCAG GAACACTTCCCCGTACTGAAG
Sequence
Wt Sequence (deletions in lower case)
AGCTGCAAAGCCCTGAGCCGGATGGCGGCGCGCTCGGCACTGCTGGAGGAAAGCAAGTGAACTTCGCAGAGACAACATTGGCTATCAGTTAAACTTGGCCTGGGAAGAACGTTTGTTCTGATAAAGCTCTCTTAAACCTCCTCCCTTGCTCTGCATTTGAGTGAGCAGGCAGGCACAGAAAGAAGCCCTCGGACTGTAAGAAATGTGGAAGCATCCTCAGTCAAGAGCAGAGCCTCGTTTGTCTGGACTTCCCTATTGCAGAGAAGCGCTGTGAATGAGATCACTCTTCCACTCAGATGTCACAGCTCAGCCAGCAGAGAATCttaagcgggggaagcccctttgcctgtaaggtatgtgggaaactcttcagccacaaatcgaatcttacagagcacgaacatttccatagtagagagaaaccgtttgagtgtaacgagtgcgggaaagcctttagccagaaacagtatgtcatcaagcaccagagcactcacagcggagagaagctattcgagtgcagtgactgcggaaaagcctttagccagaaggaaaacttgcttacgcatcagaaaattcacactggagagaaaccttttgagtgcaaagattgtgggaaagccttcattcagaagtcaaacctcatcagacatcagagaactcacacgggggagaagcccttcatatgtaaggagtgtgggaaaaccttcagtggcaaatccaatctgacggagcacgagaagattcacatcggagagaagcccttcaagtgcaacgagtgcggaacggcttttggccagaaaaagtacctgataaagcaccaaaacatccacaccggggagaagccgtacgagtgcaacgagtgcggcaaggccttctcgcagcgcacgtcgctgattgtgcatgtgagaatccattcaggcgataagccttacgagtgcaatgtatgtgggaaagccttctctcagagctcatctctaaccgtgcatgtgaggagccatacgggtgagaaaccctacgggtgcaatgaatgtgggaaggccttttctcagttctcaaccctggccctgcatctgagaatacacacaggtaaaaagccttatcagtgcagtgagtgtgggaaggctttcagccagaagtcccatcacattagacaccagaaaatccatactcattaaacgCCCTGAATATCTTCAGTACGGGGAAGTGTTCATTAGGAATTTATACCTCATAGCGCATCGTTTGGTCACTCTGGCTAAGCACCATAAATCAGCCAAACATTTAACAGAAGATGTCGTGAACACCGGAGAATTCACAATGAAGGGAAAGACATGTGTATGATACAGGCCTGCAATCAGCGAATGCAGCGGTGGAAAAGCAGAGCAGTGCTGAACTGTGAACACAGTCGTCTGCGGTCAAGAGTGGCAGGCAGGCAGGCCTGTTGTGA
This is an 855 bp deletion beginning at Chromosome 7 position 29,861,159 bp and ending after 29,862,013 bp (GRCm39/mm39).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 58397 | Fluorophore-1 | AGC CAG CAG AGA ATC CCC TG | Quencher-1 | MUT Probe | ||
| 58398 | Fluorophore-2 | AGT GTG GGA AGG CTT TCA GC | Quencher-2 | WT Probe | ||
| 58399 | GAA CAC TTC CCC GTA CTG AAG | Common | A | |||
| 58400 | GAG AAG CGC TGT GAA TGA GA | Mutant Forward | A | |||
| 58401 | GGT AAA AAG CCT TAT CAG TGC AG | Wild type Forward | A |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 58399 | 0.40 uM |
| 58400 | 0.40 uM |
| 58401 | 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.