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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
>chr18:5213099-5213198 100bp AGTGAACTGCAGCCAGGAG TCCCTTCCACGAGCACTTAC
Mut= 121 bp
Wt= 100 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
AGTGCAGGGAAATGAACCCAGGTCCTATGCAATGCAGTGTGTCCTCTTAAGTGTTGGGCCAAGTCTCTAGCCCAGGTCAGATGTGACTTGAAGTTTCAGTCCAGAACAGATACATTGTAGTAATCTATGTATTGTCATTGCAGGTGAATCAAACATCCGttctggaacaacacagagtgggaaaacattacagagtaagagtcagtttaggaccattgcaccaaaagttgtacctaaggtcctaacatccagagtgcttccctgcccttcctcaatttctgaccaagggagtctgactctaatgcccaagcctctgggcatgcccacccagaactatgctctgatgcaggtggctggccaagaggggacgttttctcttgttgctttcccaaatgttgcctcagcccagccagttcagaagcccagaatgtccacacatgaaaaccttaaactgcctattcctcgataccaaccactaagtagccaaggattaagaaagaaacaggacctgagctctcccaagagtggctccagtgaccctcctggcccatcccagctatgtcatttgacctcttccccacaagcctatcctgatcctcatcacaaacctagcctgtggaagcaaatgccaactttaaacccttctcccaccaacatcaacacagctacactgaccagtggtgttggccaaggagatttgagtcctctagtgaccaacagctgtggcgatcttgagctttctgccatgccagtgtacactacagaggactcttcttcccaacagagcctcccagcaagtatgcagaaggcaggttgtgccaggaaggaggcacccactacacctgctattgctagtgaagaactcaaagaacagttagcttctgcacagactgtcttgagcagtgcagttcagttgctctctgtagtacccaagggtaaactgcccatcctgcccttaccaagagtgaaaactacagaggtgggcaaagtggaatcagatgctgataatgcagaattgtctttatctggatgcagagcaaactgtgatgagagactgtccatcacagaagggtttgatgcagccacagaaatagccagcaagatagctgctttgcatgggtctaagcagagtgcatgtgaaagtgccttctgtcctgtcaccaaattggatctcagccacaaggcaaaacccagcagtggaataaagagaagaggaagaaaatggaaagtgccagatgagattttagcattgcagggcaaaaggaggaaatgcattattggtatgtgtggagatggcatagaaagagcaagaaacagtccccaagaagccagagaccagaagcccagggttgcttcaaggaagtatcgtaacatcatgcccaagcctgtccttgtcctgtctgctttggcacccctggcgtctcatacggctgtgctgtctcaggctcccagcagcctaggtcaagatgttctgaataatgccctgccttcaaaatgtcttggctccaagcaaagtgacagctctacccctaagcccagctctgtactccgaaatggattctctggcattaaaaagccctggcacatgtgtccagtctgcaactaccactttcaattcaaacaccaccttctagaccacatgaatacacacaccaacagacggccttatagttgcgggatctgtcgcaagacctatgtgcgccccggcagcctgagtgcacatatgaaacttcaccatggtgacaaccaccccaagaaactagtgtgctgtgaattttgtgcaaaagtatttggtcatgtccgagtctatttcggccatctgaaggaggtgcacagggttgtgatcagcacagagccctcccccagtgaactgcagccaggagacacaccaaaaaacaagaacagagatcctggtATGCAAGGACCTGACAACTCACTGGAGAGGTAAGTGCTCGTGGAAGGGAAGCTGACAGGAAAGTCTGCTGTAATATGAGAAGGAAGACCAACTCGTGGATATTTCATTCCTCCCTAGCATAGGGAATAAAATATCCATGGAAGGAGTT
This mutation is a 1756 bp deletion beginning at Chromosome 18 position 5,213,148 bp and ending after 5,214,903 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 42679 | AGT GAA CTG CAG CCA GGA G | Wild type Forward | A | |||
| 42680 | TCC CTT CCA CGA GCA CTT AC | Common | A | |||
| 42681 | TTG AAG TTT CAG TCC AGA ACA G | Mutant Forward | A | |||
| 42682 | Fluorophore-1 | ACC AAA AAA CAA GAA CAG AGA TCC T | Quencher-1 | WT Probe | ||
| 42683 | Fluorophore-2 | CAA ACA TCC GAT GCA AGG A | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 42679 | 0.40 uM |
| 42680 | 0.40 uM |
| 42681 | 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.