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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
>chr6:128981251+128981385 135bp CCCGAAAAACTGGATTGGAG ACCAGCTCCTTCTCGTTGTC
Mut= 117 bp
Wt= 135 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
TATTCAAGGCAGAGAATAACAAGAGGGAATCTCCAAGTAGGAACTTCAAAGGTGAGGCCAGGAAGGTACAGTGCCTTTGACCATGAGCCATGAGTTTAGATACCAGGCTCAACTCTATTTTGAAAGTATTAAATGGAAAGTTCCTGAAGTAAGAAATTTATAGGATTTTAGTACCACAATATTCAGAATAGTGCAATAAAATCTTGCACTGTCCTCTTAAGTATTTGAAGTCATCCTTTAGTGCAAGGTGTCTGCACCGTATATACGACCTACCCAAAAATTCTCATAGCAATCTCAATTATCAGGCTGGGTGTCAGTAGGTGTCCCTACAGAGTGCTTGctgctgtagcaatccccactgtagtcaatggtcatccaaagctcagaaagtgatgctgttatagaagtgcacttcctgggagccctactgacagtgagcatatgagagagaatgggacacaggcccacggtgggaggccttttagttaaaggcacatctcactcaggagaggattcctacagatcagttaggaaagctaccatcagattcacacctcacagctgagctcaggagggtgtgccaaaacgagagaaggcctgcttgccatgatccattgtattctctacattttagcaacaaagacagaacagatcctaatcaacaagacctatgctgcttgcccgaaaaactggattggagttggaaataaatgtttttatttttctgaatacacaagtaactggacattcgcccagaccttctgcatggcacaagaggcccaactagctcggtttgacaacgagaaggagctggtaagcaatgggcagggattggtttgtctgtctgttctgttgaatattatattgccttgagatagagagttacagatgaggcctgaggaaggatcccacccaagtacatggagacatagggaatgtgagtgtgcgccatttgctgatacttgacttctgactggagccctgagatagtcaagaaacattctctcatgaagttctcatagtcagctggaaggtcaaatatgccattttactgggatacctggtgaccatgagtgttttcccatatgctggcatatgttgggtacagaaggagacaactgataataactgcagtggaaggttaacccagaactgtccagaccacagaggaatgtgaccctcagttacatcctcctgttatctctagagaaaggtgtggagtggagagactccaggatcatctgaaacaaatagacacatgtattcttgacttttttgtgttttatgacagaatttcctaatgagatacaaggcaaattttgattcctggattggactgcacagagagtcgtcagagcacccttggaagtggacagacaacactgagtataacaacatgtatgttttcacgatgtttttccttctattatgttcatgtgttgtgatatgtgtgtgtcgtggctatgagagatggaAGTCAATGTCATGTGAAGCCAACTGTACTGGGAAGAAAGAAAAAAAAAATGAACCCTTGCCTGGAGGTGTGGCACAGGGGTAGAGAGTGTGTATAAATGCAATATCCAATCCCCAGAAAGCTCTACACACCCACAAATTTAAATACTTCAGAGGTTTTCCTGTTTATTGACCGTCATTTTCAAAACCTTTGCATCATGTCATTTTACTCAAAATATTTACCATAATGATGGTGTCTGAGAGTAGCTATTGTTTGCTCTGGCTCCAACTTAAACATTTCTGTTGTTGATAAATGTCCTGTGAGGGATATAGACAGAGCCTTAGATGGGCAGTGGGGGCTCTGA
This mutation is a 1135 bp deletion beginning at Chromosome 6 position 128,980,911 bp and ending after 128,982,045 bp (GRCm38/mm10). In addition, there is a 9 base pair insertion (ACAATGCAC) at the deletion site.
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 53000 | CCC GAA AAA CTG GAT TGG AG | Wild type Forward | A | |||
| 53001 | ACC AGC TCC TTC TCG TTG TC | Wild type Reverse | A | |||
| 53002 | CGA CCT ACC CAA AAA TTC TCA | Mutant Forward | A | |||
| 53003 | TCC CAG TAC AGT TGG CTT CAC | Mutant Reverse | A | |||
| 53004 | Fluorophore-1 | CTG GAC ATT CGC CCA GAC C | Quencher-1 | WT Probe | ||
| 53005 | Fluorophore-2 | CAG AGT GCT TGC TGC ATT GTA GTC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 53000 | 0.40 uM |
| 53001 | 0.40 uM |
| 53002 | 0.40 uM |
| 53003 | 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.