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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
>chr1:133659500-133659612 113bp GGAGACATAAAGAGAATGAGTGTATG ATGGAAACGCACCCCAGA
Mut = 116 bp
Wt = 113 bp
Fam = Mut
Hex = Wt
Sequence
Wt Sequence (deletions in lower case):
AGGTTCTGGTCCGCATTGTGGAGACATAAAGAGAATGAGTGTATGTACTCTAAGTGTACCAGTTTCCTCAAtctctcctggcagaagatgcagtacttttggtgatgctgggattctggggtgcgtttccattaattcaaacacagacgaagacgatgtggtagagggaaagatggtggcagaaggagcgaataaagagacaaaattgcctgctaaaaagaaaagaaagaagggtttgcgaattaaggggaaaagacgccgaaaaaaattgattcttgccaaaaagtttagtaaggatttggggtctgggagacctgttgctgatgcccctgcttcgttagcttccggtgcccctgagcaagatgaagaaagtctttttgagggcaacatagaaaagcaaatctatctgcctagtaccagagccaaaacctccattgtgtggcacttctttcatgttgacccccaatatacctggagagcaatttgtaacctgtgtgaaaaaagtgtcagcaggggtaagccaggcagccatcttggtacatctactcttcaaagacatcttcaggcaaggcattctcctcactggaccagggccaacaagtttggagtcactaatggggaggaggactttaccttggatttatcgttgtctcctccttctcctggaagcaatggaagctttgagtatattcctactgattctgtagatgaaaacagaatgggtaagaagcgcgataagtcagcatctgatgccctgagggcaaaaagagggcgctttctcatcaaaagcaacattgtcaagcatgccttaattccaggaacgagagccaagacatctgcagtttggaattttttttatactgaccctcagcacatctcaagagctgtgtgtaacatatgtaaaagaagtgtgagccggggtaggccaggttcccacttaggaacttcaacacttcaacgacatcttcaggccacgcatcccatccattgggctgttgccaacaaagacagtggtgctattggaaacggattagatgagactgaaacagagagcagtgatctcctgaatgatacgatgcctggagagaagtcatcaggcagccaagatttaacagctgaggacctcagtgactctgatacagatgaacccccttgtttagaggttgaaaatagatctgagagtcctattcctgttgcagaccaagataatccggtgcacgcacaagagagagaaacgacgacacattgtgaaaatgcagccgcaaatcaaataagtcaggcagttattcagatgattgtggaggatctgcatccgtacaactatttctcaaccccagcctttcaaaggtttctacagattgtcgctcctgactatagattgccatcagagacttactttttcaccaaagctgtacctcagttatatgattctgttagagaaaaaattttcttaactttagagaatgttcaaagccaaaagatccacctgactgctgacatatggactcatgacccttccactgactatttcattgtgactgtacactgggtctctttggaaactgcatcttctcccagtaatggtgggactcccaattttagaaagtgggcagtactttgtgttacaggtttagccaaagactgtttgataactaacattttacaagaattaaatgaccagattggtctgtggctttctcctaatttccttacccctagcttcattgtttctgacaattcttctaatgtggtacatgcaatcaaaggtggtggctttacccatgtgccatgcttcctgcattgtctaaatatagtgattcaggactttttctgtgagcacaaaagtattgagaatatgttagtggctgctaggaaaacctgccatcattttagccattctgtcaaagcccgccagatactacaagaattccaaaatgatcaccagcttccatggaagaatctgaagcaggatgaaactggccactggatttctaccttttacatgttaaaatggcttttagaacattgctactctgttcaccatagtctgggcagagccagtggagttgtacttacctcccttcagtggactctaatgacatacgtttgtgatattcttaagccatttgaggaggccacccagagagtgagtgtgaagacaacaggattgaatcaggtgctacccctaatccaccatctcctcttttccctgcagagactcagagaagattttcaagtcagaggtattactcaggccctcaatctggtggacagtttatctttgaaacttgaaagtgatgccttactaagtgccatgctcaaatccaagcattgtatcttggctactttgttagatccttgttttaagaacagtttggaggacttttttcctcaaggtgctgatttagaaacttacaaacagatccttgcagaagaggtttgtaattatatggaatcttcgccaggggcctgccagatttcaagttctgaaacatctggccctttagttaggttaggaactgattcgtttacttctataaaagaaggcacctccagtgcaggttctctggatagctcagccgcaggcagtgttgctgttgggagcaaaagctccttgctccccgctgcggtagcagtagtagatgagtacttcaaagagaagtattcagagctctcaggaggtgatgaccctttggtttactggcagaggaaggtgagcatatggccagctttgacccaagttgccattcagtatctgagttgccccatgtGTAGTTGGCAATCTGAATGCATGTTTACCACAAATAGCCACTTTCATCCAAAGCAGATCATGAACATGGACTTTGA
This mutation is a 2700 bp deletion beginning at Chromosome 1 position 133,656,861 bp and ending after 133,659,560 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 52240 | GGA GAC ATA AAG AGA ATG AGT GTA TG | Common | A | |||
| 52241 | ATG GAA ACG CAC CCC AGA | Wild type Reverse | A | |||
| 52242 | TTC ATG ATC TGC TTT GGA TGA | Mutant Reverse | A | |||
| 52243 | Fluorophore-1 | CCT GGC AGA AGA TGC AGT ACT TT | Quencher-1 | WT Probe | ||
| 52244 | Fluorophore-2 | AGT AGT TGG CAA TCT GAA TGC ATG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 52240 | 0.40 uM |
| 52241 | 0.40 uM |
| 52242 | 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.