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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
>chr19:5893009-5893101 93bp CATGGAGCTGAACACCAAGA TGGGACATCTTAGACTCATCCA
Mut = 91 bp
Wt = 93 bp
Fam = Mut
Hex = Wt
Sequence
Wt Sequence (deletions in lower case):
CTTCCCAGGGAGTCATGGAGCTGAACACCAAGAAGAAGCTTCACGCCCTgtccctggctgagaagatccaggtgctggaactcttggatgagtctaagatgtcccagtcagaggtggcccggcgcttccaggtctcacagccccagatctcacggatctgcaagaataaggagaagctgttggcagactggtgcagtggcacagccaaccacgagcggaagcggaagcgtgaatccaaatacagtgggatcgatgaggctctactgtgctggtaccacattgcccgagccaaggcctgggatgtaacggggcccatgttgctccacaaggccaaggaactggccgatatcatgggccaggatttcgtgcctagcattggctggctggtccgctggaaacgtcgaaacaatgtgggctttgggactcgccaggtccttgtccctctgttccctcctgaagctcctcctgcagttctcccatctcaggcccagccacctctttctcttaaagacttctcaccagaagatgtttttggctgtgctgaagtacccttgttgtatcgcgcggtgcctggcagagtgtttgaatgtgatcggttgcaagtactgttatgtgccaacagcagaggcacagaaaagcgaagggtatttgtgggtgggctccaggcggccccaagatgcttctttggggtgagcagtgaggcactgcctacctcttatcaccctgacctagccattccctggtcagagtggctggcacagtttgaccaggacatgggacagcagggccgacaggtagccttgctactggcttctggagtggtagaggaatgggccagccttcctggactccatcacgttcgactcctgcctctctctgcctccagcaccactccttccctgcctggctctgtgattttggcctttaaggcccattaccgtcaccgtctactgagcaaactggctgccatgcagagtgggaaagagggcacctccctggcggaggccagggctagcatcacagttttggatgctctgcacatggtggccgcagcttgggccaaggtacctcgccagctcattttgagcagctttgttcaagaagggctcgctccgggcaaaacaccactgtccctggacaaagacactgagatgtcaccagtccctagtgggctgagccaagaggagttttctcactttgtggacctggaggatgaggacccagggcctagagtgtgcaaagaggagacgggtactgaagacagtgggagggaagaagatggctttgagcccctgcccaccaaagccgatgccctgcaggcactgtgcaccctgaggaggtggcttgaatgcaacagtgcctctccagaactctttgagaaattctatgactGTGAGGTGGAGGTGGAGCAGCTCTGCTGTCTGTGAGGAGGCATAGCTGCCTATTAGTCTCCCTCTCCTGTTTCCCATGGAAACGCCTCTCCAGAGACAGACTGGCTCTTTCCTGTGGAAATAG
This mutation is a 1343 bp deletion beginning at Chromosome 19 position 5,891,723 bp and ending after 5,893,065 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 52235 | CAT GGA GCT GAA CAC CAA GA | Common | A | |||
| 52236 | TGG GAC ATC TTA GAC TCA TCC A | Wild type Reverse | A | |||
| 52237 | TAA TAG GCA GCT ATG CCT CCT C | Mutant Reverse | A | |||
| 52238 | Fluorophore-1 | TGG CTG AGA AGA TCC AGG TG | Quencher-1 | WT Probe | ||
| 52239 | Fluorophore-2 | CCT GTG AGG TGG AGG TGG A | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 52235 | 0.40 uM |
| 52236 | 0.40 uM |
| 52237 | 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.