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= 98 bp
Wild Type = 116 bp
>chr3:89248340+89248455 116bp TCTTTGCCTTCCTTTCTGG TCCCATGACTTAGGAAAGAGG
Sequence
CACAGGTGACTCTAGGCCATGGGATCATGACTGAAAGATGAGTGCAGCTTGTCCAGAGAGACAGCAGATGCCTTGGCTACAGAAGGCTGGAGACCAAGCAGGGCGGTGGCCTAGAATGCTCTTAGAAGCATTATTTCCTCCTTGCCACAAGAGGGCGCTGCACCCAAGGCGCGTAGAGGAAGAAGTAAAGCAAGTCCACGTTGGCCCCGACGCCTGCGT[acagacaaccgcgccggctttggctcccctctttctccagggacacggattggaccacggacggggcgtgatgggtaaccgcaccggcgggatgcccgggctgctctagcgggagtgggcgttggggtggtcctcttttctccgtagcctcggtaaagctgacgtgtcctgggaactagcatttcctccggacaggcgggaacttgggtgtgcagggccgggtggttcctggaggtcgctgtggggtgggggctgggtcacttggaagtggttggtgagccaagctccggagtgaatctgcctgaatcttagcctgactctttttctcatttatagtagtcggtaccgggacctccttggcgctgtcctccctcctgtccctgctgctcttcgctggcatgcagatctacagccgccagctggcctccaccgagtggctcaccattcaggggggcctgctgggctccggccttttcgtcttctccctgactgtatcttcctcgcgctggaaggggaggcgggggtgtatgaacagaacatgggcgggagtgggtggcaagggctttgcagtccctctacttcctatcttgttgcgtgagtttctccttaattacatcacccaggccttcaataatctggagaatctggtctttggtaaaggcttccaagcaaaaatcttccctgagagtaagtgaaactcaatagttaactggactcttttttttttaaacctctcccaaacatgatgccgaatttccctgtgcttagatgataaaggagaaatacatttgcttaacacaatcttgaagacagaagatgctttcagattaagcattttattggagctggaagagaagcacaaaaaggggggaggggaggggaggggagggagaggaaaagaaagaaaaaggccaacctggtctgctctcaggacaggtttcaagtgaacagtcaagcctgggctgatgtctgataggcatccaggagaaggtgttctttgtccctgtgagagaagaaagcaaaaccaagcccactgagggattctgatgtggaactgagcccgcactattgactgctgctggaggagattgtgaagtctgtgttcactgggatgttaccaatagcaggacccctgacagaaggagaatagagaataaccgctagagctcactctctttctctgcatccgcttgtttctctcttcctttcttctcctcccttctgtagcccacactggccttgaacttcctgtgtggctgtgaatgactaaacccttgacttgctgatcttcctgcctccacttcccaggtcctgagattagcactacctcatgtagtttatgaggtgctggcgattaagcccaggtccttgaacatgctaggcaatctctctcccaatggaggcacatctccagccctgatagatcagtttttgtgtgtgataattcttgttagatggttcatatagagatctgatggagaatagggctcataaaacaaagtcggcatcaggaccgagccacttggaaagccacatgctgggaaaagcaagcagttgtggccccgtgaaagactcacccagaaaccccgaagcccgccattgttgctgtcttcacggccctgttggaggtttccagagcttccgcccagcaatgcccagagcttctgtctcttccactctgtcccatgtgtttcttatatgctgaaagaaattgttggaaggcaaaaaccttgtctttcagccagacagtgatggcacaggctcttaatcctagcactagaggcagagggaggcaggtctctgtgagttcagggctagcgcgatctacagagcaagttccaggacagccagagctacacagtgaaacctgtcttgaagtgagaagaaaaaaatcctcttctttgccttcctttctggctccagctggacaagtggacctcttagccccttccaccctctcatccgtatcctgtcctcttagagaagccataatcctctttcctaagtcatgggaacacgcctgctttccgcccttctagaacattttatttattgatttacttttgaggcgggatctcactatgttgcccaggctggccttcccactgtgtagcccaggctgtccctcccttggattcctgggcttacgtgatgcttcctgccttatcctccggactggcttgctgtgtaggcatataccactgcgttttcgctttcttctgtgtatgttcgggggttgggggaccaggaagttaaatgaaacgtggagccaagctagtcaaacaaaacgccattcatatcttcccttggtgtcctgaacaagtgttcaggcacaaggcatttgaaatccctgcttttattaaggcatacctattttgtttgtgttgtttttctagtcaggatttctctgtgtagccctggctgtcctggaactccctctgtagaggaagctgacctccagctcacagagatctctctgactctgcctctagtgctggggttaaaggtgtatatccccatgccaggcaggacgtacgttttcaagcaggttcctcacagggttctagactgctcctacttttccgttgctcttttgaccgccttctgattgaccctcttcctgtctccccagttctcctctgcttgctgttggcgctctttgcctccggcctcatccaccgagtttgtgtcaccacttggtacgtatctgacagctgggcaagcggtgcaacaacaccatgtcttgtgggaccagatcttcctatggtgttaatagtcccttcccttcccacc^GGCATATGGTGGGAAGGGAAGGGACTATTA^]ATATGCCACCTCCTCTGTCCCCTCCCCCCCATTATTTTTTTCAAAGCTTTTTTTATTTATTATATGTAACTACACTGTAGCTGTCTTCAGACACACCAGAAGAGGGCATCAGATCTCATTACGGATGGTTGTGAGCCA
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 46251 | AGG AAG AAG TAA AGC AAG TCC AC | Mutant Forward | A | |||
| 47717 | GGA GGG GAC AGA GGA GGT | Mutant Reverse | A | |||
| 47718 | TCT TTG CCT TCC TTT CTG G | Wild type Forward | A | |||
| 47719 | TCC CAT GAC TTA GGA AAG AGG | Wild type Reverse | A | |||
| 47720 | Fluorophore-1 | CCC TTC CAC CCT CTC ATC CGT | Quencher-1 | WT Probe | ||
| 47721 | Fluorophore-2 | CGC CTG CGT GGC ATA TGG T | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 46251 | 0.40 uM |
| 47717 | 0.40 uM |
| 47718 | 0.40 uM |
| 47719 | 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.