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Stock No: 037617
Protocol 43276: Probe Assay - Ramac<em1(IMPC)J>
Version 1.0
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
Mut = 140 bp
Wt = 137 bp
Fam= Mut
Hex= Wt
>chr7:81767325+81767461 137bp CTCTGGGAGGTATGGTCATGT TGGAGTAGTAACAAAAGTCCCTGT
The WT Reverse primer (primer 62665) anneals over the nucleotide sequence containing mouse genomic variation rs241068838.
Sequence
Wt Sequence (deletions in lower case):
GGGCCTGGCTTTTCATACATGTGCTGGAGAGCAAACTCAGGTCCACAGTCTTACAGGACAAGCATGTTACCAGTTAAGCTATTCTCCCCAGTCTCATATATGTCTCTTCTAGACTGGTGGCTCTGGGAGGTATGGTCATGTATTTATTGTTCATTTTAGTATTGCAGGCTCCAATAgcgtagtgtagtcaagaactgttgaagtgaatgcctacaatcttaagtttaaaatacacagggacttttgttactactccatctaaattgttcacttcttattttttcatcagctaagtcagtgtaatttttttaaatacttacagatcttaaaaattgtacagaatttcaaaatgagtgatacttctgaagaaattccaaattttgaagagatgtttgcaagcagattcacgaaagatgacaaagaataccaggaatacttgaaacgccctcctgagtcccctccaattgttgaggaatggaatagcagagctggtggtaaccaaagaaatagaggcaactggtatgtgttatttttgcaggataaatgcaaagggtgatctgggagaagagagctttaatacagttgctcacagagccagctagagtagcaacataataagcatataaatacctgcttttttgtgttttattaaagccaggaatagtgatagaaaccatactttcattactggggaggctggggcaataggattatgagatctaggccagcctgggccaaatagtaagaccctgtctcaaactaatgttttctttataaatatactttataaaattggagttttgtgcttcatatcataaagaaggtattgttttgattactttagaaattttataaaatggggtggaaaattaaacgtagtaattcaagacaataaatgatgctgatgtacgcatttaataggtgtcagtttgatcacagtcaggtttattccttgcctctatcctgtaaaagacgtccagtgtgtgatgctgggagactcacttaaatcttaggaaaatctgacttgtgctgtaccacaaataccacttgtgctgtaaagagcctccattactgtgtattatatcaagaactcaattgcactaggattgatttcttgtttttatttagtaaagggaagttaatattcttgtcttcttttggtaggctgcaagacaacagacaatttagaggtagggataacagacgaggatggccaagtgacaatcggtcaaatcagtggcatggacggtcatggggtaacaacaactacccacagcagagaccagagccctactatcaacaacagtacacacagtatggccacaaccagcggcctccatatggttactattgacagaaatgtcggcagcttttaacaaagaccttttattttgacaacgtgctaaatgcttgtgtgtcttctgttggtcatagtttttcattctattttaaagaatgggttattaattgattagaacttgagacttggaaaaaagtaatcttattagaaacctgatgattgatgggaataaatttattaagtgaggattatttgacttcttttacatactgttttgttctatgatttggtagtgttttgagttttccttatttggactctagggattctgtattacagaaataaaaatttttaattaaaatgctggcttttgctgtatgaaagaatagcatgctcacacgtgctcacctctgaagtgtttcagaggctaggactgtcagtctgtgaaaacacacccacaattacttaaagtaattgaaccacagttcagttgaaaggtaaaacaggtatgtcgcttctgtgtttgtatgtgatcatggtgtcttgtggtttataagtgagtcagctccagaactgctttggtcctatatggaagggctcattttctgtgcagcctcctgaggcggtggagggtgggatggtagcactcaacattttgtatatcccaagatttttgtaaacttacaacaaggaagacatggatgtgcgctgtaacacaatactcatgtaaatattgttgcattttgattgttttggtattatgtattgatttttatgagattattttcattttcaaatgcttaccttcgatgcttcctcatacagttatagtaaggctgtcacttgcttcagaacaatcattcttgaaatattttcatgataacagatttattcaaataaaaaaatttaaagtccttagtatattatgtgtgtaccaagttgtgaaagaatgaataaataaattgcattaaaatgtaggcaactttaccttttagttttgacagcccatttatTTATGTGGTAATGACTTAATTGGTTTTTCATTATGCTGTCCTTTATACAGTGAATAAGAGTTAACTTCCCAAGTTTGCCTTTTCTCTTAGCTCCTTGAGATGGACTCTCCCAGGTTCTCTGTAATCAGAATGTATCAAGTCCCTCTTTTTAGCCATCCACTCAGGTAGCCATCTCATTAAGAAGG
This mutation is a 2137 bp deletion beginning at Chromosome 7 position 81,767,381 bp and ending after 81,769,517 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 62664 | CTC TGG GAG GTA TGG TCA TGT | Common | A | |||
| 62665 | TGG AGT AGT AAC AAA AGT CCC TGT | Wild type Reverse | A | |||
| 62666 | GAA AAG GCA AAC TTG GGA AG | Mutant Reverse | A | |||
| 62667 | Fluorophore-1 | AGT CAA GAA CTG TTG AAG TGA ATG C | Quencher-1 | WT Probe | ||
| 62668 | Fluorophore-2 | CAG GCT CCA ATA TTA TGT GGT AAT GA | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 62664 | 0.40 uM |
| 62665 | 0.40 uM |
| 62666 | 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.