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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
>chr5:30959413-30959501 89bp GCGGGAAGCAAAAGTACAAC CTGTGAGGGAACCAAAGTGG
Mut = 89 bp
Wt = 89 bp
Fam = Mut
Hex = Wt
Sequence
Wt Sequence (deletions in lower case):
GCTCTAAGCAACAGCAACAACAACCAAAGAAACAAAAAAACAAAAAAAAACAAAAAATAAGAGCTGGAGGTGCGGGAAGCAAAAGTACAACTGAGAGAGACTAGGATTTAATgtgacttccgggctggcctgacctttgcccactttggttccctcacagcattttctgcagctagagctgatcaacgggtgcctgagcgcttccttgctgcactctcatgacacggagacacgggccaccatgaatttggcgcttgctggtgacattcttgctgccggacaggatgcccagtgtcagcttcttcgtttccaggtccatcaacagaagggcagtaaagcggagaagtcaggtgaagagctttggaaaaagtcgctctaattcttggtctccaaaaacgagagattaggtgtggggcagtggtcacattgaagtctgtacttgggtcactagaggactggtgttagttaatacagatgctcaggtccatctgagggaggtggtgcacagctgtagtctcagagcttaggagtggaggcaagaggatatttaaggctagcctggggtgcctagcaaatccctgtcttaaaaaacaacaacaaaggaatctggttcctggcaaattgtttgtgttgcaggttccaaggagcagggacctcgacagagaaagggggctcctccagcagagaagaaatcgggagcacaagttcaccccgaaggggttgaactcaaagtaaagaatttggaggcagtacagacagacttcagcaatgaaccgctgcagaaagttgtgtgcttcaaccatgataacaccctgcttgccactggaggaactgatggtcatgttcgtgtctggaaggtgtggttttaggggcttgggggggtgggtgagtgttagttgcaataacaccaaaattgtgagaatctgaatatgcattctgtgaaacttgagtggccattggggtaagagggatggggatttcagcgttctcgctgtctggtctgtaactgtcttggctgtaggtacctagcctggagaaagttctggagttcaaagcccatgaaggggagattggagatttgactttgggtcctgatggcaaggtaaggaggtggagatgtgggatgctggtgcagaaaggcagagcctattgtgtgccttgcacagtacatagatgtcctaactgtccatctttgggatctttactcctccccagctggttacagtgggctgggactttaaggcctctgtgtggcagaaggatcaactggtgacacagctacagtggcaagagaatggaccagcctcttctaatacaccataccgctaccaggcctgcaggtgtgcaggtcacagggtggctggcaggcacacctcagctgggaaatgggaaaagacagaaggagctggccatggctggagccttgttggaggttgttccaggtgtgaccagggtgcagggcacaaacatgagggctgacaggaactgaaagccttttgcctggcctccaggtttgggcaggttccagatcagctcggtgggctgcgactcttcacagtgcagataccccacaagcgcctacgtcagcccccaccctgctacctcacagcctgggacagttccaccttcttgcctcttcggaccagatcctgtggccatgaggtcatttcctgcctcagtgtcaggtgtgaagtcctactgcttgattggatggtgggcgcttgagggagctggggaatgagtcctgactcagtctctggaggaccactgttGTGGGGAGGGAGTTTGCACAAGCTTCAGAGCAATGAGCCCTCTGTTGTGTGTTACAGTGAACCCTCTGTTGTTGTATGTATGTTACAGTGAATCCTCTGTTGTATGTTACAGTGAATCCTCTGTTGTATGTATGTTACATTGAATCCTCTGTTGTGTGTTACAGTGAATCCTCTGTTGTGTTAC
This mutation is a 1660 bp deletion beginning at Chromosome 5 position 30,957,801 bp and ending after 30,959,460 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 51203 | GCG GGA AGC AAA AGT ACA AC | Common | A | |||
| 51204 | CTG TGA GGG AAC CAA AGT GG | Wild type Reverse | A | |||
| 51205 | ACA ACA GAG GGC TCA TTG CTC | Mutant Reverse | A | |||
| 51206 | Fluorophore-1 | ACT TCC GGG CTG GCC T | Quencher-1 | WT Probe | ||
| 51207 | Fluorophore-2 | ATG TGG GGA GGG AGT TTG C | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 51203 | 0.40 uM |
| 51204 | 0.40 uM |
| 51205 | 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.