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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
>chr4:135418208-135418312 105bp CAGTATGGGGTGCAGAGC GACCACAGCACTGGAAAGTGT
Mut= 120 bp
Wt= 105 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
CCATGCCAAGAACACACAACTCAGTTAATAATATTTATAAACTATAAGCCTAGACTGGGCAGATCGACCACTACACTACTCTAGTCCCCAGCTGTGAGATCCCTTGCTACTTGCGGCTCCTCTAGGCCACGTGGCTCTCCTCCATCCTCCTTTCTCTGTGCCTTCATCTCATCATCCTTTCTTCCTTCCTCCTCTTCTCTAAAACTTCCAGCCCCACCTTTTTCttccatggcccaatcacaggctctagccattatttgaccagttaaaatggggagcagggttcccatgttatcacctgagtagtgatccgctcctcgctggggccaggattagcatcaaaatacaaacagcaccaaggcaatctacaataggtagcaactctgaacaccagtgagaaaggagccgcagcgcccacgcgagcaacatagggagtttgtctggcggcttccccttagcaggtctcccgttaacaggaatattctgtcttccctcccggtgcaggagaggtttgaggccctgttcaccctctacgatgaccaggtcacattccagttgttcaagagctttcgcagagtgaggatcaacttcagcaagcccgaggctgcggcaagagcgcggatagagctccacgagagtgagttccacggacggaagctgaagctttacttcgcacaggtaatggccgttctgcgcctgcgcacacagcctgctccagttcccgctccagcacggggtcagaggtctgtgaggtcagcagtcacgtgagccagggctgccgtgctttttctgactttacacatacgtcatttcatgtattttaagagcacattgagcctctgttcttgtttctctgagacgaacacctaagggattcgtttttctggcgattttgctcagctagggctctgtgagggaagtcctgatacttcgaggttggcagattaaacactgtgcatctaaaatggcaccgagaacatgacatccgtgggaaaacagaacaaaaccttcaagggtcatcaagatggcccagggggtgaaggtgcttgccaccaagcctggcagcccgagtttgatcccaggaactcatccacgggtggaaggaaagaaccaacctgtgtcctctgaggaccacatatgcagttttctctcttctgagacagtagtgtgtttgttagtcagcccttcccagcgaattagttactgggatgagacactgtgaccaaaagcacccaggagacaaaaggtgtatgtactttacttataatgaatcaccattcattgagggaagccaaggcaagaactcaacctgggcagaaacctggaggcagaggccatggaggggtgctgtttactggctcctcggcctactcagcctgctttcttttttttgttttgttttttggtttttgagacagggtttctctgtatagccctggctgtcctgaaactcactctgtagaccaggctggcctcgaactcagaaatccgcctgcctctgcctcccgagtactgggattaaaggcgtgtgccactgtgcctggcttcagcctgctttcttatagaacctagaaccacaacccaggctggtatcattcacaatgggcagggccttccccacattggtcactaagaaaacttcctgcctgcagtcaggtcttctggagacattttctcagttgggttcctgtctcttgatgactaaagcttgcatcaggttgacatatagtagccagcacacccactcacaccactagcaaatacctgggagagtcagctgtaaaggagaaaagtctcggcttgtggtttgcaggtttcagtctgcatgtgattggcacttttcctgtgagcctgctgtgcagtagcacataggggcagagcaaagctcttcacttcgttcatgggaagggggaagagtaaggggttggggttccactgtcccttagggtatgtccccatgactaaaggcctccctgcctcctgaaggctcccagtttgacctctcaggggagcaagcctctatctactatgtagagcccaagggtcacttagagcccagaccacagagtagcacgtttatcaagggtccagggcctgtggccacttccagtccaccacctggaaggtcacagacagtttgagagacagttttaatcacccctccaagaaagtaacaattaccataaagttggaaatgaaagccctgtggtgatggtgcaggcctttaatctaagaactggaggcagagaccgtgagatctgtgagtcaggcctacagagtgagttccaggacagccagggatacacggagaaaccctgtctcagaaaaagaaaagaaaggacagctgctcacaagcacgcctttccctgcaggtgcaggtgtccggggaggcccgggacaagtcctacttactgccaccacaacccaccaagcagttcctcatctcccctcccgcctcaccccccgtggggtggaagcagagtgaagatgcgatgccagtgatcaactatgacctgctctgcgctgtctccaagctgggcccaggtactgcattccaccttcgctctccgagtcctcggacattgctgttctgtgtgttggagactgtgtgcagtatggggtgcagagcccagcaacaccagcaccgtccagtgggcggtgtggccacaccagtctgagttcacacTCGAGCTGTACACTTTCCAGTGCTGTGGTCCTCAGCCAGTTGCCTAGCCTGGGTTATCTGAGTGTGTTCTAAGGATTAAACGCTGTCTGCAGCGTGATAACTTTAGCCATTCAGCCAGAAGTTAATATAGGCGGTTAGTGAACATCCTCACTGCTTTCTCTCTGCAAGCCAGTCAGCACAGTGTCTGTCGTTTGGCAGCTGCTTTGGGTGACAGTGACAATGACCTATCACCCTTCCAAAGTTCTATCTCTC
This mutation is a 2462 bp deletion beginning at Chromosome 4 position 135,418,238 bp and ending after 135,420,699 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 48100 | CAG TAT GGG GTG CAG AGC | Wild type Forward | A | |||
| 48101 | GAC CAC AGC ACT GGA AAG TGT | Common | A | |||
| 48102 | CTC TCC TCC ATC CTC CTT TC | Mutant Forward | A | |||
| 48103 | Fluorophore-1 | ACA CCA GCA CCG TCC AGT | Quencher-1 | WT Probe | ||
| 48104 | Fluorophore-2 | CTA AAA CTT CCA GCC CCA CC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 48100 | 0.40 uM |
| 48101 | 0.40 uM |
| 48102 | 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.