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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
>chr3:105815164+105815302 139bp TCTGGACTCTGGACATTCTACA TTGTTCACACCTTGTCACTGAT
Mut= 134 bp
Wt= 139 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
GATGTGAACTCATTTATGTTGAATGGTCTAGATGATGATAATGCTTTGGGCTTGTTTCTTAGAGTGTAGTCTGGACTCTGGACATTCTACAAAATTTAATTTCGAGTATAAGCAGAGCATAATGACGACCACCCCATAAGAatttggatagaaggacggcagcaagtgtgggtcgggggagccttgatcagtgacaaggtgtgaacaaggcagaggctttgggctgacaaggaacagtcgccagacttcaaagcttgtgatgaggctgacagcattgaggagctgtgtagagacttggctgtcagccctcctgatagtcacagatagggcagctttcatgcagagttctgtacacaccctgggctgccttcagggctgcatggatgtgggtagagtcatctcagcagccgcaggctcctcctgctatacccacttcctcgtttgcaaactgcttcacttctgagagaccagggccctttccagtagctttagggatggtaagttgtaagtgattcttgctaagtattccttttatgctcttaagtctcatcagagagataagaaatcagggcccagataaacaggtgcctgtgttacattcactgtaagtgagtctgcagtgaagctcacgtgaacatgctggaggcaaaggcaagttccagggttcaagttcagtaggattctaacagcaaacaatcacgccgctctgcttcttttgtttaaaacaaacaactagaaaatgcctccttattaggtaggtactgtacgctatgattgttaaatcgccatcctgaacatttcaactgtttgtgtccccatgcagctgtccatgacccaaacttggaggaggacaagtgaatgcatctcataaaggagaaggtaatataatcaatcgctttatctaacatttctacctgggcaaagatggaagtttaattttcaaggtacacggatactctctgttgacactctaccacacggcagaatgctctgctgatctcctctgccctgagttaatttgggacctttcctctcagagaaacagtaaacaatattaattttcacagcattgtgaagttggtgacgtcgctcaagcatgatgacagctggtcagtaggtcacaagttctcctcgttgttattcccacagactgggaagccaaggcggagaaggacatgtgagactgggcaggcttcagctgtcatctgtgggaagtggcttttggttgaccagcggtacttcccagtgtcagcacgtgtgttgtgtcggcagctcagaccttgcgctgaggggcccctgtcctcagccacccttcatcttgggaaaactctctggtgtagagcagggaggtagggaggcagggaggcagggaggcagggaggcagggaggcagggaggcttcctagtgctaactcaacttgtttaaaccattatactttatctaaataattgcattgaatacttcgatacaagtaagaacacgcaggagagaagtaggagaggctgccgtggaagaagaagaatatgcaggagagagaaggagagctgccatgtgctaaagggccaagagaacgtggcccagagggctgctcagttgggtcaagagcagccaagatggaacatagaaactagtaagtagtaactcagagttaacagtaggaaagtggattctaacagcatagagagtaggcagttacccagctatttgtgctgcctaaggcatatatatatatatatatatatatactttgatacaaagccctactatgtctgtgttgttatccctgttttaaaagaggactccatcgggttctcgtagttaggaagctatgaagtcaaactttaaaacccaagggttcctgactgcaaagtccactttctttccagttgccttgctgaagtgcccgctctgaatgaagaagaactgggtcctgcagtctgtccacaggaagaagactgtctgtgagcacagtctccatgttcactgtctcatgccgtccctgatatcatggaggaaatgaatggtcatttaagaatcctcattggctaaagagctggtggtttaaatgaatgaatctaatgcaaactgtgaagggctggtcctggtattaagaagatggacgattctgtgaagggaatgtgctaagacttgtttgttgaaaatggaattattaatggcttgtcagtaaagttaagaatgccttaaatcattcctctcttactctattcatagaagaaataaaacagatgaatttataaagggtttattgtctgccagacattattttttatatttatatgggtcatttaattctgcaatgatcctacttggaagatatagttatttattacagttgaaagttcgggggaagaacagaaaggttaagaaacttgcccaaggatacaaaggatggaaatggtaatggggcagcctgaacttccaaatctggacagctattcactgtcccttctgctcctgtctaatagaagggaagatcagacagaggtatgaaggtctgtgtctatatttagaaaaaaaattcttctttctcatttttttattcctagagggatagccactttttgcaatgctgagccatacTAATGGCAAGACAAGTCTACTGACTATCACTCTTAAGTCTCTCACCACATATCCTCATCCCTCTCTTTCCTTCTCAGCATGACTCTAGCATCCTCCTCTGCCCTTGGGAGTGAGGGGTGACTGTGGACTAA
This mutation is a 2461 bp deletion beginning at Chromosome 3 position 105,815,236 bp and ending after 105,817,696 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 43201 | TCT GGA CTC TGG ACA TTC TAC A | Common | A | |||
| 43202 | TTG TTC ACA CCT TGT CAC TGA T | Wild type Reverse | A | |||
| 43203 | AGG GAT GAG GAT ATG TGG TGA | Mutant Reverse | A | |||
| 43204 | Fluorophore-1 | AAG GAC GGC AGC AAG TGT | Quencher-1 | WT Probe | ||
| 43205 | Fluorophore-2 | CCC ATA AGA TAA TGG CAA GAC AA | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 43201 | 0.40 uM |
| 43202 | 0.40 uM |
| 43203 | 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.