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
>chr17:53452568-53452713 146bp TTTTAGGTACAGCCCAAGT AGCTTCTCAGTCAAACAGGTTGC
Mut= 162 bp
Wt= 171 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
TCTGTAGACCGGCTGGCCTCGAACTCAGAAATCGGCCTGACTCTGCCTCACAAACGCTGGGATTAAAGGCGTGCGCCACCACGCCCGACTTTACCGGGATGCATTTCTGCTACTTGTTTGTTTACTATCTTTTCAATTGTCCTCTTGTTTTATTTTAGTAGCATTTGTAGCGTTTTAGGTACAGCCCAAGTTTAAAATACATTCCACTCCttaaccaggctttcttgaccctgaaatgatcaacgctttttttccctctaaggcaggaaaagttaagccagttggatacagagttgcaacctgtttgactgagaagcttcccagggtgagcacgtcccagtcttcctgtttgttcctcaaatatgagttgtagggagactagattctcagctttctttatgaaagggtgcacagatgtaacgtgtctagttttgaatctttactatattttggcgttatctattgatccatattcatccaagaaaatgaaaatgaaaataaacatttttagtttaccaaaaattctaagagtcaatgtatttcagtaggaattatgggatacttgtaacctggatgtttggggggggggggagcgggaggagggtaggggatgtctgtgtgactgtaagtcacatgtgcgcaggtctccatgggagccagaaaacggcatggcatctttggcactagagtgctagatggttctgtgcaccatgtgggtacagggaacccaactcagggcccctggaaaagcaacaactgctcgtgtaactacttagtcttttccagtcccctacattatagtcttaaaaatgttttaaaattaatttttattttatgtgtatgagtgctttgcctgtacttatgactgttcaccaggtacatacctggtgcccacagaggtcagaatggatcatcagatcccctggatctggagtttcagagcaatgagccaacatgtgggtactgggactgaacctgggtcctcttgcaggagcaacaaacgctcttatgcacttagccatctcttcaggcccccaaactataatatttaaagtatttttttccaccagtagacacagtttagatggtgtgtcctagttttcagatgcaggaagtcatcactggctagggaagctgagttgatattttgaaactcatgattcatatgtctctttgtctcactttgcccagctaatgactcccccagaggcaaagaagtatttcaacttcagatatccacctgctggagcagaaagagtattttatggcagagcaaatgacccccaaattgcaccttatttgacacatggactaagatctaaaatctcaataccggtaaacctttctcttttttttcctcctacattaatcattcatttacgaagttagaaagttgtacaaatcaaggtgtaagtgaaattcataattactatgggtattttccttcctcattctggcttctaaaaacactagtcttgggcaatgggttgtgttttcaagtaactaaggataggcgaaggaagcagcagttcaaacccagagtttactgatcctcagatgggatgaggctgcgacattcccattttcccagtaaagaaaaatgttggaatatcttattagccattaagcgaataggcctgctccttttgcccaaggccctacagagagcatatatagatcttgagtggatcagaaaatatttgatgctgactgctgggatataactgtcctctaattgcttatttatctaccttcctacctttccttctatatttgttatctccttttatttatttcagtttattaagtttatttatttagttatttactggtggggctaaagctggaactcggggtaatctgtattctgggtaaacactactgttgagacctatcatcaaccctctatctttttgtttaaaaaccttttcccacattttgtgcgtgtgtgtgtgtgcgcgcgcgtgtgtacatgtgctacagcacctgtgtgggggacagatgagaacttgggacagtcagttctttccttctatcttgtgatttctggagactaaactcagatcatcagccttggcagcaagctcccttacctgctgagccatcttggccattaaaaaaaattcattgattaatttttagcgtgtgtgtgtgtgcacatgcacgcatgtgtgcacgcacgtatgtgttttatgtgtgggcatgcatgtagggacttagaggacaactttgtagagttgattccctctttctttacatggtttctggggatcaaagttacactggcaggctcttgtggcagtatttttacttgctgatccatgcctcagggccttttctgtttttgagacaatgtactatgcccagcatctatctctcttgtctttctcttcctgtctatctctcccctgctttttgtttgtttgtttgtttgtttgaggcagtgatttttctacatagccatagctatcctggaacttgctttgtagacaggactggccttgaactcagatccacctgcctcttcctccacagttctgggattaaaggcatgtgccacaccatgcccagtgctttctctcatttttatcttttgttatttgaagtttttaactctgtagtccaggttggcctcaatcttcctgcctctgttacttaaggactgggattacagtactgtgtcattaggtctaatctgggttttatatgtttgagaaagttggtggaaatgattttatgttctgtgcttgtcatacgtgttcttgagtatttgcatcaataacctggaaactccttccataagtcgttagcaccactgtaggaaagttaaatttatctgtctgtgtatgtatgtatgtatgtatgtatgtacatatgtacatacttaactatctatttacttaatgctggggattgaacccgtggtcttgtacctgttaggcaagtgctctattgctgagctacttctctagtccttttttattttctgctttgtggttagaacttacagcctcatgagcactccactactgaaccattctccatctggtcttgcaatctttctgtagtttcatgcctgcctggagcttagtatcctggtacttcagttcccccaagagctgggatacagtcctagctgaaagttctgcatctgctttgttaaacttttcagtactgatttcattgtcagttcattggaagatgagcacagccaatgatgatgggggaaatagcactttaatcgtattttcttctcattagagttttagttttcttacaaatactggtttcgatacatgaacattctataatttctttagatgggatcattgataaacccacagcccattaccacgtttcaacagaaaattaaagacaaaaaagaatctatatattttagccatcaacgagcaccattgggaaaatctcatgatcaaacaccaggattgccaaaaggaatggatgtgatcaacacaacattagggacaccgactatcagaggtagtaaaaataagggacaggaggaggtgggaaatacgtcttatgaaactggattaaatatgtcactgatatattttctgttgatgatgtgaaggattgttagtgatgaaagatgaacatagtcttcacacaaggtcagtggtctgcttgggccattctctccttctatcttgtcctgtTGACAATGACATCACATCCTCTCTGTCCCCTAACTATAATCTCACGTTTGCATTTTTTGCTTTCAGTTTTCTGCTTTTATTTAAATCTATACTTTTTTTTCTACTTAGTGGGTATCTTGCCATGGTACCTGAAGAGTGTCGCACAGAGAGAGATGACTGAAATACTCCAGGTACAAGTAAAAAGATGGTATCATAGCAGGATCTCTCGCT
This mutation is a 3470 bp deletion beginning at Chromosome 17 position 53,449,206 bp and ending after 53,452,675 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 48285 | TGG CAA GAT ACC CAC TAA GTA GA | Mutant Reverse | A | |||
| 48286 | Fluorophore-1 | ACC CTG AAA TGA TCA ACG CTT | Quencher-1 | WT Probe | ||
| 49191 | Fluorophore-2 | ACA TCA CAT CCT CTC TGT CCC CTA | Quencher-2 | MUT Probe | ||
| 49281 | CGT TTT AGG TAC AGC CCA AG | Common | A | |||
| 49282 | ACT GGG ACG TGC TCA CCC T | Wild type Reverse | A |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 48285 | 0.40 uM |
| 49281 | 0.40 uM |
| 49282 | 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.