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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
>chr11:99591312-99591424 113bp GCAGCCAGCCAGGGATTAT CAGGCCATGGTGTCAGAAGT
Mut= 118 bp
Wt= 113 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletions in lower case):
GAAGATTATAGTGAATATGGGTGATCCAGGCTCATGTTTCCAATGAACGCTAATGACTACTTCACTGTAGTCAAGAAGTCTAATTGAACACCAAATAAAATCAGCTTTTTACGGGCTTTCAGAAGTCTGAACAAGTAACTCAGAAACAAACAATTATCATTTAGAGAAAAACTACATAAACATAAACAAGGAAGACACTTTGGAAATTAGGGAAACAGCAGCCAGCCAGGGATTATAaaaagactggagtggcactcaccacacaaattcaaaaaacttctctaagcaaacaaaccttcagcacagatcaacttctgacaccatggcctgctgcgctactagcttctgtggctttcccacatgttccactggtggcacctgtggctccagctgctgccagcccagctgctgccagcccagctgctgccagcctagctgctcccagtccagctgctgccagcccagctgctgccagtccagctgttgccagcccagctgctgccaatccagttgctgccaaccttgctgctcccagaccagctgttgccagcccagctgctgccagcccagctgctgtggaactggcagtggccaggagggtggcagtggagccgtgagctgccgtgttagatggtgccgccctgactgccgcgtggagggcacctgcctgcccccctgctgtgtggtaagctgcacacccccaacctgctgccagctgcaccatgcccaggcctcctgctgccgtccatcctattgtggacagtcctgctgccgcccagcctgctgctgctactgctgcccacccagctgctctgagtctaactgctgtgagcctacctgttaagacccaggttgctgattcccaaaggatcttgaattttgatatggcttgtgtttggggcacaaatacttggccttactaaacatttttttccatctaatactggaaaggctttgatatcctctatgaaaataataaaatacaaacttgatccccaaacaagaaaatcaatttaccttgggattgtccctattttccaaggcttggtgctgtcaaggccatggaagattcttctacacgaagatagttttcattcttcttccattgttctgtaaattcctcttataaatgaaggctaatgtatccaaaataataaattatatctgttggcatatcaaaacttgcttctttttatttagtgtgcaaataatagatacttcatggagatccatctaaaagatttctgagtttgattctatacacttatctattttgtgtggggagtggggcatgtaagtagagatcagtctctccttccaccttatgattctgaggactgaactccagtcatcgggcttagaggcaagtgcctttacccactgagctgccttgtcagccctgtttttttatattcttcttatacctgttaggaaatgggatgaattccaacagtggttagaccccaaaaaagttgtagcattgccctggtcttgttttattttgctgccgtcaataaacattgtgtccaaaaacatggggagcaaagaggttgtttggcTTATACTTCCAGAACACAGTTCATCACTGAGGAAAGTCAAGGCAGAAACTCAGAGTCAGGAACTTAGTGGCTTGTTTTCTAGCTCACTTGTGGGATCAATTTTAGCTAGCTCTCTCATAAGGCCAGCCCCTCCTCAATCAATTAATAATCAACATAGTCTCTTGTAGGGATAGAGACAGTCACTTGGTTGAGGGTCCCTTTCGGGCTATCACAGGCTGTGTCT
This mutation is a 1322 bp deletion beginning at Chromosome 11 position 99,590,083 bp and ending after 99,591,404 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 64388 | GCA GCC AGC CAG GGA TTA T | Common | A | |||
| 64389 | CAG GCC ATG GTG TCA GAA GT | Wild type Reverse | A | |||
| 64390 | TGA TCC CAC AAG TGA GCT AGA A | Mutant Reverse | A | |||
| 64391 | Fluorophore-1 | CAA ACA AAC CTT CAG CAC AGA T | Quencher-1 | WT Probe | ||
| 64392 | Fluorophore-2 | AAC TCA GAG TCA GGA ACT TAG TGG C | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| 64388 | 0.50 uM |
| 64389 | 0.50 uM |
| 64390 | 0.50 uM |
| Glycerol | 6.50 % |
| Dye | 1.00 X |
| Kapa 2G HS taq polym | 0.03 U/ul |
| 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.