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
>chr7:35794645-35794735 91bp TGTCATTGCTATTGGGGACA AGATGAAGACGGTGGGTCAG
Mutant= 133 bp
Wild Type = 91 bp
Sequence
Wt sequence with deleted region in lower case
CAAAGTACTTCCCAAAATTCAGTATTCCTTATAGTAGGATGTGAATATATTTTCAGAATTTCAAAAATTTGAGCAGAGAAGTGTGATTTTTCGTTATCTACAGTTAAAATTTGGCATAAGAAATAGTTTATCCAGAGTTACATGAAATCATGTTGGCTTCACAGACTGATGCTTTCCAGTAGCTCGAGGTGACTCCACAGGCCCCAAGGATGGCTGTTTCTTTAAACAAAGAATCATGTCTCTTTAGTTCTAGAATCTCAAGGGTTTCTTTAAACAAAGAGTCATGTCTCTTTAGTTCTAGAATTGCAAGGGATTCaagggacattagttctagaatctcaaggaggctgggtgacaggataaggtgttaaagtcttacccaaggaacagggtctttatctgtctctaggagaccgtttcaaaaaggaattctgagctgtttaagtctattagggacagttgggtgcattgataggaacctcccaacttagaagatatttaaacatttcagtcagacaccagagttgacattatcccataacccatgctctctgctgtgcactgtctctagatatggaagaaagcagcagcattgccatgttggtgcaagagattggagaaccggaggctgtgttaactgctgaaggagtcctcagcccttcctcagaagttgaccaacaaagaaaagctaaagcagatccattggtccacgttatccagaagctaagcaagatagtggggcatgagaagtcacaaaaatgtctactgattgggaaaaagcgcccacgtccaagtgagacagcaaactctcttgaaaagctagagaactgtgagattccagctaaagccactgagtcccctgctgctggggttaggaaaaccgagatgtcacaggcaagttccactcttgcttcaaatgatgggaaggctatgtcctaccagtgtagcctttgtaagttcctgtcaccatccttttctgtgctgaaagagcacgtcaagcagcatgggcagcagcatgacgtgatgctcatgtgctcggagtgccacgccacctccaggagccagcaggagctcgaagcgcacgtggtgagtgagcacgagaacagtgccagtagccaggcccggtccagcccctccggtcagggagccacagaaagaaagagtgagaccatggtagacatcccagtgaacatgggcagtccacagacccatgctgtccagagtgcagccatggcagagtcgggtcggaggaagtggtatgcatatgaacagtatggcatgtaccggtgcttattctgtagctacacctgcggccagcagcggatgttaaagacacatgcttggaaacatgcaggggaagttaactgctcctacccaatctttgagaatgaaaatgagccccttggccttctggcttcttctatgtctgctgcacctggtggggttgatgcagttgtcattgctattggggacagcgagctgagcatccacaatgggccatctgtacaagtacagatttgcagctctgacccaccgtcttcatctcctttagaacaaagcacagaggagggagtacacctaaaccaggcagtcacgctggacgctaacgaggaagaaatgctggaggtgatgtctgattccgaggagaatctctttgctgatagtttgctatcttcagcacagaaaatcattagcagtagcccaaacaaaaaaggtcacgtgaatgtcattgtggaacgtttgccaagtgctgaagaaaccctcccaccaaaacatttcctcattaatgccgaaatggaagaggggaagagcccgagcccctccgaagcccagactgggtgtgtaggagcaggaaatatgtaccatgctgataaatgtactgttgatattggggggttgatcatcggctggagcagtgcagagaagaaagacagtgagctaagtaagggcctggctcctgatgagaatgccccaccaggacggagaaggacaaattctgagtctctcaggctgcactccttagctgcagaagcccttgtcaccatgccgataagagctgctgaactaacaagagccagccttgggcactatggggacataaaccttttagatccagacactggacaaaggcaggtcagtggtccattggcaacatactcaaaaaaaatcatgtctcctcttaagaattctacagatggagtaacaagttttaaccaaagcaattccactgtggtagctctcccagagggaaggcaggaattgtcagatgggcaagttaagacaggcatcagtatgtcccttcttaccgtaatagaaaaactgagagaaaggacagaccagaatgcatcagatgatgacattttgaaagagttgcaggacaatgcccagtgtcagcccaacagcgatgggagtttgttggggtccaacgtggtggagtacatcccagatgccgagcggccctaccgatgccgcctgtgcaactactcaagtggcaacaggggctacatcaagcagcacctgcgtgtccatcggcagaggcagccttaccagtgtcctatctgtgagcacatagctgagaacagcaaagacttggaaagccacatgatcaaccactgtaaaaccagaatacaccaatgtaagcagtgcaaagaatctttccattacaaggtgagcggcggtctcaagggttggcagggactttatagctgtagtccagccaccccagtttgtttatcaatgaaaatgagttccagagaagtttggtaatttgcccaaaggtacccccaacaaattaccattgaagataggtctggaagctaagtctccatggttgctgtccagagctttcccttagtctgatgccgtgccctttgcagcctcagcgcctgttgctttcATGTTTTCCTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGAAGCACGGGTTTTCACTTAGAAGGGAACATAGCGTAGACTGACAGGCACGTGTCTGACGGCAAAGGCTAACTGTGCATCTGCACACAGACCAGTATTCCCTGCACTCATAGCAGCTGCTACACAACCAGCAAACAGTGGTCTCTCACTGCAGAAGGGAAGCAGCCTGTGAGACCTGCTAGGAGGCAGCACAAGAGAAATGATACCATATTTGCTATTGCTGGACACTTTGAGGGACTTTGTTAGGAACCAACTCCAAGACCTCACACCAGCTAGGCAAATGCTTGCCTACCTGTGAAGTGCATTCCCAGCCCTTGTTTTTGTGAGAGTCCTCCTGAATGTCCCAGGCAGAA
A 2580 bp deletion beginning at Chromosome 7 position 35,793,294 bp and ending after 35,795,873 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 45148 | TGT CAT TGC TAT TGG GGA CA | Wild type Forward | A | |||
| 45149 | AGA TGA AGA CGG TGG GTC AG | Wild type Reverse | A | |||
| 45150 | Fluorophore-1 | TCC ACA ATG GGC CAT CTG | Quencher-1 | WT Probe | ||
| 46376 | TTG CAA GGG ATT CAT GTT TTC | Mutant Forward | A | |||
| 46377 | TAG CCT TTG CCG TCA GAC AC | Mutant Reverse | A | |||
| 47215 | Fluorophore-2 | CAC GGG TTT TCA CTT AGA AGG GA | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 45148 | 0.40 uM |
| 45149 | 0.40 uM |
| 46376 | 0.40 uM |
| 46377 | 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.