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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
Mutant= 140 bp
Wild Type = 164 bp
>chr15:58550058+58550221 164bp GCAAAACTGCTGACTAAGATCCAT GGTGATGGTGACGGTGATCT
Sequence
Wt Sequence (deletions in lower case; insertions with carrots ^g^ (g insertion):
CAAGTCTGAAGTTTGTTCTTACTTGAGATATATTAATTTGCTGTTACGTTATTTATTTTTATTTTATACTTGCACACAATGTCATATATACGTATATATGTATGT^t^atgtatgtatatatatacatatatatgtgtatatatatatatatatatatacacacacacacatacatatttctctttactttaatagagcaaaactgctgactaagatccatgatggagagatcaaatctcaaaactatcaagtaagtgatgggttcctcctacccagcttccctggtcaggagactgaagagcaatgagaagcgagtggtgtgggcactcttttgtgttgtagatcaccgtcaccatcaccgaggcccgtcagctggtgggcgagaacatcgacccggttgtgaccattgagattggggatgacaagaagcaaagcacagtgaaggaagggaccaacagtccattttacaatgaagtaagtcacagagcatcctctgggcccacagtgaaggaaaggaccaacaatccattttacaatgaagtaagtcacagagcatcctctgggcccacagtgaaggaagggaccaacagcccattttacaatgaagtaagtcacagagcatcctctgggcccacagtgaaggaagggaccaacagcccattttacaatgaagtaagtcacagagcatcctctgggcccacgtcctcttcacaggtgaggagaagagcgtagccactcggtgCTTCTTCCTGGTTCCATCTAAGGTCACAGCCCAGTTTGAACTCTTTCCACACAGCCCCAAGGCATTGATAACAAGGCAGGAGACAGGAGAGGAAAAGAGGCCCACTTCCTTACCTCACAAAAGCTCACTGGCATTCTTAGTAAGC
This mutation is a 635 bp deletion beginning at Chromosome 15 position 58,549,969 bp and ending after 58,550,603 bp (GRCm38/mm10). There is a single bp insertion (T) at the deletion site.
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 67500 | GCA AAA CTG CTG ACT AAG ATC CAT | Wild type Forward | A | |||
| 67501 | GGT GAT GGT GAC GGT GAT CT | Wild type Reverse | A | |||
| 67502 | TTT ATT TTA TAC TTG CAC ACA ATG TC | Mutant Forward | A | |||
| 67503 | CTC TCC TGT CTC CTG CCT TG | Mutant Reverse | A | |||
| 67504 | Fluorophore-1 | AGA GCA ATG AGA AGC GAG TGG T | Quencher-1 | WT Probe | ||
| 67505 | Fluorophore-2 | TCA CAG CCC AGT TTG AAC TCT TTC C | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 67500 | 0.40 uM |
| 67501 | 0.40 uM |
| 67502 | 0.40 uM |
| 67503 | 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.