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 = 88 bp
Sequence
Wt Sequence (deletion in lower case):
ATGCTTATGTGACTTAGAAACATATATTAAGATGTGTCTTAAAGATATGCATGCTGGTAGCTTTTTATAATCTCAACACTCATGCAGCAGAGGCAGGGGGATCACAGCTTTGAGACGGGAAGGGAGGTGTGTAATAGTCAAGAGTctcactggcagctgtcctcagaggagcccaccaagcagagcccccagctggtccagccccgtgtaaatctcagaagtgtgtctactcagtttcattaatcaacagaaaacatcctttttgtccatacactggttctgatatttctcttttcttttccccaagattgctccagaccatgtcgtccctgctccagaggaatgctatgtgtatagtccattgggctctgcctataaactcaagagttacactgaaggctatggtaaaaacaccagcttagttactatgtgagtaaactacctcttggagcatgaattaaactgcgtgaatgaagataactggctgtttacttctgtcttaattaaaatgcttgaggagcctgacctcatagtgttcctgctattactctttcagttttatgatttggaataccatgatgggaacatctatactaagtattccttggggcataaaacaggtaatataacttttgagtacacttttatctaaaaatatcttaagatttattattttttatttttaattatgcatattagcaagtctgagagtgggtgtttgcatataaccccctgtgcccatgggggtcagaggcgctggaccttcctggaactggggtttcaggcagttgtgagccacccaacatgggtgcagtgaggcaggcttcagtgctctgaaaggccagaatgcattcagaactgctgagccatctcttcaacccccgaggatttttccatacctataatcccaacacttaggctaaggaactcatggattgtgagttcaagaccagtttgggctacatagtaaattttaagctagcctgtgtaaataaatgaataaccgcactggaaaagaaaatgatcactatattttgttttattacttacataaaattctctttagaaagttgatggttctgtttatttttccactagcgtgtttttgtcagaattgtttagggtatcagtgtaattgcatatgaagttattcttacacaacaaaaggaacctgactttttatttttaaaaaggctggatttaccactgggatgtgtgtcatcgtgctgatgggccttctaacactttactgctgctatagagtggtgaagtcacggagtacgataagtaagtagagacaggtttttgtgggaaataatgcactctcctatgaggcagatcctactgtgtacattctatatatgtgtctccttctaccttttctgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgagggtaaaggacagcttgaaggtatcagttttctcctactgtgaggttctctggaatagcagctccctaaCTTAAGCTCCTGCTGGCCCCAGCTGGCTCGCTCTGAAATTCTGGGCTGAGCATTCTTTGGAGAAGACAATTATATTATATTATATTATATTATATTATATTATATTATATTATATTATATTACATTATATTAGGGCGTGTGTCTTTTAGGGCCTAGACAGGCATGGTTTTCCTTCAGCCATATTAGCAAATGCAA
This mutation is a 1350 bp deletion beginning at Chromosome 13 position 112,689,111 bp and ending after 112,690,460 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 68406 | GCT GAT GGG CCT TCT AAC AC | Wild type Forward | A | |||
| 68407 | TCC CAC AAA AAC CTG TCT CTA C | Wild type Reverse | A | |||
| 68408 | CAC AGC TTT GAG ACG GGA AG | Mutant Forward | A | |||
| 68409 | GCT CAG CCC AGA ATT TCA GA | Mutant Reverse | A | |||
| 68410 | Fluorophore-1 | ACT GCT GCT ATA GAG TGG TGA AGT C | Quencher-1 | WT Probe | ||
| 68411 | Fluorophore-2 | CAA GAG TCT TAA GCT CCT GCT GGC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 68406 | 0.40 uM |
| 68407 | 0.40 uM |
| 68408 | 0.40 uM |
| 68409 | 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.