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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
>chr15:75981236-75981348 113bp TTGAAGAACCTGGAGCAAATG TGACTGGTTGACCCGATG
Mut = 120 bp
Wt = 113 bp
Fam = Mut
Hex = Wt
Sequence
Wt Sequence (deletions in lower case):
GGGCACTTGGAAGTTAGTTCAGTGTCTTGAAGCTGAAGTTCTGCCTGACCTGCGACACTGTAACTGAAGAATCATGCAAGGATATAAGAAACCTTAGGGAATGAATGATTGAGTCAAGCCGTTGccagagcaacagaatgggatgaggtcgaggcactgcaaccaagttctgtctccatagaggttgttcaagctggacatcaacattcctagagctacgcaccaggatgcgaaagctgtggtctggagggaacgcttatatacagattggccacggtgctctctgatccttaatcctgcctgctctgaacctcataatcagaagacacacccttgaacctagggtgcagttctccagaattttcgggttaagaagggagagtaactgagctgggggagcgggtggagcacagcgctctcatgagtccagttggcttgggcctcaagccatggcgcccaagaagaagcgcggacaaaacgcggggcgcaagcaggaaactacagagcaaccgctctcggagcgcgcgcagtacctacagcgcgagtacgcgttgctctcagaaagcctggtggcctgtgagcagcggatcgacgaggtgctgcaggacaacgagttcctgaacagagaggcgcagcgcctgcgcgaagagaaccggctttatgtcggttacgtgagcgcgcacgctctgcgctgcgccaacgccgtcgtgcgcgtcgaagaccagaaccgcatggatctggcgcagatccggtggcagcgagcggagctagcgtccttctaccacggccgtgaggacggggtgcgcgctcagctgcaggaaatgaagaagcgcgcggaaaacatgacgcagcgggtgcaggagctccagccctacaaggccagtgcgcgcgccccccgaggaccgggcgggctccagagggatgggcgcgctccctaccggtgcgctgatcggcgccccctgctccccaaccctcaccccaccccatgtaggagctgcagctggagcagctggcgcgaatccggaccctggagcgcgagctgctgcacatgcgcgtggagcacacgcagctcctccacggggtgaagcgacgcttcctagatgagaagacggctttcgagcgggaggcgcggctgcaagtgcagtccctgacgcgccgctccgagcgggaggccgcacgttccctcatttcccacgcacaggccctcaaggcggacaacgggtatctccgacaagaactcatgagacttctccagaggacccagttactgcaggacatgcgacagcagctgctagagcagcgggagcagctgcgccgggagcatgtggacttgaagaacctggagcaaatgcacggctggttgcaaaggggccccggggggccaccgctctggcagccaccccaaagcctccaacccagcttgcgcatcgggtcaaccagtcacgttttaaaagcccaggctacgccccagtcagttccaacctcccggggccaatccatggatctgtcacaccgaccctccaagactccatctatattgtccagtgagtctcttccgggcctatctcagaaggctgggtcagtgattgctctccggagtccatcacatccaggatctcgagtctcatctctgactccatcacgcaaggactcgcgtgtctcatccgcgaccccgtcacgcaagggctccagggttccatccatgacctctggtaccttgcggtcgcgcgagggctctcggatctctccacagccctccttgcgtgaggtctctccagaaattgacacacctgcaaagtccagttccaagcttcccacagccctgttggaagatcaggcccctctcagcccacagttggaggaggcagaaaacgctgaagacgacacagaaactgtcttggagcaagcctgaacccacacgaggaggcaggccagtagggcatagggtggaggacaaaccttggaagatttcttggtgcatagatatgttaataaaagtgttaaataagctctgtgacgctctgacacttgcctgggtcgatgcccttgtatttcaagtgagcagaggcctgctattcgagctacagcagagctggttgtatccttggctgctggaattcgatgttcttccaccaacagccagcaaggcaggcaggcttagggaaagttgggtagtgtaatttcagcagcagagggcagattctgttctcagcacagggagaagttactgggcttttccaatgctttatataatacgcacacacacatatgtatgtaggtatgtatatgtatatatgtatatacattccaatttagggaacactgcaagaattattaggaattcccattgccacccatctagatgaacagggatgcatgttatatatttggtactttcagccatccgggattctgttggagacagttatgccccttaattttaaatatatccctgtgtttactgaataggactttgacctgttttcttccggtgctgggtgttgaacccagggctacctcctgagctacagcccaagccctgtcccaactttcttcaagacatgattaaaattaggaaatgcaccttggagaaaatactgtaaactaaatcatactggccaaattgtccctgtggtgtctgtagctagctatggatcttacctgggtcacatcttacattccattgtgctggtctgtcctttcctttatagaaagggtctgtataggtcaggctggccttgaatccaagagatcctcctgcctctgtctcttcagtgctgagattaaaggcatccaccaccacctcagccccgttgtcttgtctttttttttttaaacttctttttaaagatttatgtatatgagtgcactgtaactgtcttcagacacaccagaagagggtgttgggtcccattacagatggttgcgagccaccacgtggtcaccagggattgaactcaggaccactggaagagcagtcagtgctcttaaccgctgatccatctctccagccctctttgtcttaatgtacaagaccagtcGTTAGAGCACTCCTTAACTGAGTTTTGGAGTCTGTTGGCTTTGAGCACCTTTCCTGTCCAGGAAGTCCAGGGCAGTTGACACCTCCTGACCCAGG
This mutation is a 2917 bp deletion beginning at Chromosome 15 position 75,979,638 bp and ending after 75,982,554 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 52088 | TTG AAG AAC CTG GAG CAA ATG | Wild type Forward | A | |||
| 52089 | TGA CTG GTT GAC CCG ATG | Wild type Reverse | A | |||
| 52090 | TGA CCT GCG ACA CTG TAA CTG | Mutant Forward | A | |||
| 52091 | AAG CCA ACA GAC TCC AAA ACT C | Mutant Reverse | A | |||
| 52092 | Fluorophore-1 | CCA AAG CCT CCA ACC CA | Quencher-1 | WT Probe | ||
| 52093 | Fluorophore-2 | AGT CAA GCC GTT GGT TAG AGC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 52088 | 0.40 uM |
| 52089 | 0.40 uM |
| 52090 | 0.40 uM |
| 52091 | 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.