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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
>chr14:55632770-55632860 91bp GTGGTGGGAAAGGAATAGCC TGTATACCCCTTCTCTGCCTTC
Mut = 111 bp
Wt = 91 bp
Fam = Mut
Hex = Wt
Sequence
Wt Sequence (deletions in lower case):
GAAAACTAAAGTTTTTTGTGATTTATTTTTCATTTTTTAATTTTTTGGTTTTTCTGAGACAGGGTTTCTCTGTGTGGCCCTGGCAGTCCTGGAGCTTACTCTGTAGACCAGGCTGGCCTTGAACTCAGAAATCCGCCTGCCTCTGCCTCCCAAGTGCTGGGATTAAAGGTGTGTGCCACCACTGCCCAACTAAAACCCTAAAGTTTTTGAGAATGGCTGAAAATGATCAGCTAAACCCAGATAGGGAAAACAAGACTGAATTGAATTCAGCCTGTCCAGTGGTGCTGACCGAGCCAGATCAcaatgcccccctcctttcctcacccccagctgtcggtggtcttatggggtctgccttaggaaagaccgtgcacatacctgcattctctttctggccactccttgggcctggggcattgtttgcaagggaagggtggtaaaaggtgtttctatatgaatggacctcctctttgtaggcattctgtgagtgtcagcttggcccagctctcagccaccatttttcgaaaggaaggcctacaaggatggcctcagttcatgaaccttcttcagcacagcactcatagttcccatagccccgagaaagaggtactggcactgccctgttggcaggaaggagggacagaggctggatagaccacgagaccgtctcttctcccctgctcctgtacaggtgggacttctgctgctgagtgtggtggtgtcttcccagcccgaggcattccatgcccaccagcacgagctgcttcagcttctgaatgagacgctcagtgacgtcagctttcccggagtgctcttctactccctgcgcactctgactgccatcgcccgctatgtcaggccggatgatgtggtaagtaagacgctgcctgtatctctcggtttcgtgccctcgctttcagaagtcttggcccaagctgctttcgagttttattccgcctcccgttcttcctaagggttgagggcatagcgtgttagctgggcctggctggctccttagatggaggctttgaggaagcctccagctcctggcttctgggatgagaccacctgcgctttatgtgattacgctcattcctgtgtcttccagtctcttgcacggatgttggtgcccaaagtcgttacggcactgaggactctgatccccttagatgaggtaaggacagacgggaggggtctccttttggaaggaaaatggaggcacaagcaccatggtcccctagaggagcgagtaccctgtgacttacttgtctgtctgcaggtaaaggcctgtgaggccctggaggccctggatgaaatgctggagacagagttgcccatcatcaacccccacctctctgaggtcctcacgttttgcctggaggtgagcccggggtgggtgctgctcctgctcgctggtttgccgttaagttttcttgcttgtttctgattcttgtcggtgtctgtataggtggctaagaatgtggcccttggcgaaccactccgcgtacgtgttctctgctgcctcactttcttggtcaaagtcaaaagtaaggtacgttccctctggggaagttaccccatcctgctgtctccgccattagttgtgtttgtaggcttagtcctgctcgagccctggcccagccttctctccctgcctaggagacggggagtctctggctggggtccttgtggttgggaggctgggattttcccatgagcctgttctgggaagatgctcaaggctagccctcccctcttaggccttactaaagaaccgcctcgtgcctcccttgctgcacgcccttttccccctcatggctgctgagccacccatgggccaactggatcctgaagatcaagattcggatgacgatgacttggagatcgggctgatgggggagactcccaagcactttgctgtacaggtgatgtattggccttctcgtcactgtcacccaatactcagagaaacagacaccttaaggggaggaagtattttcatggcttcggagatttccatttactgtggtgggaaaggaatagccaaacagtggttcaggcctcggcaaacCAGAACCATTCACGTTATGGATAGAAGGCAGAGAAGGGGTATACAAGGAGGAGACAGGGCAAGATAGCTCCCATAGGATGTGGCCCCAACTAAGCTCTGCCTTCTGCCTTTGGCTCCTCCTAGTGACTGGCATTAGATTAAGAGATTAACTCATCTTGAGGTCAGAGCTCTCCTGATCTAATTGGCTCTGAAAATATCATCACAGTTTGTCTCCTAAGCACTTCCCAGTTCAGTCAAGTGGATTATTAAAATCAGCCATCGCACCCAGCTGACCTGGTGGACACCTGTAAGCTCAGTCCCCAGCCCTTGGGAGGCTGAGGTAGAACTGTAAGCTTTGGGCAGTCTGAGCCAT
This mutation is a 1170 bp deletion beginning at Chromosome 14 position 55,632,815 bp and ending after 55,634,584 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 48949 | TGT ATA CCC CTT CTC TGC CTT C | Common | A | |||
| 48950 | GTG GTG GGA AAG GAA TAG CC | Wild type Forward | A | |||
| 48951 | CCC AGA TAG GGA AAA CAA GAC | Mutant Forward | A | |||
| 48952 | Fluorophore-1 | TTC AGG CCT CGG CAA AC | Quencher-1 | WT Probe | ||
| 48953 | Fluorophore-2 | TGA CCG AGC CAG ATC ACA G | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 48949 | 0.40 uM |
| 48950 | 0.40 uM |
| 48951 | 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.