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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
>chr1:10047381+10047512 132bp TTGTGGAGTTCTCCCCTTTG GGCTGTTGTTGGCTGCTT
Mut= 96 bp
Wt= 132 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
CTACCTGGCTGATGAACTTCAAGATCTGTCTGTCTGTGATCTCTCAGAGTTTGGGTTACAGACACACGCTGCTGGGCCTGGCTTTTACATGGGTGTTGGTTTGGGAATACAAGCTCAGGTCCTCAGACTTGCACAGAGGGCACTTCACTGACTGAGCCGTCTCCTCATCCCCACTCTTTACATATCATGCATATGTAAATATTACATGCAAGTACAACATCACATAACTAAGAACTACATTTGTTTAGTGTctccagcctatcccctagcctcctggcccaatagcagaagcttcccaattttgtttttcactcttttctttctagtggctggcccagtgtacttaatagtaactcatttttaagaggatgaattaggtatcagaacagttgttacagtatgtaagtactttacacttcaatctctgtgctgtgtaattttttctaagttatttagtatgtcaaataagttaaattgtacattttgtggagttctcccctttgatttgtaaatatgtcttttccagggaaaggcgtcagagaaactctctgaaaacagtaaaatattaatctctatggctaaggagaacattccaccaagcagccaacaacagcccaagggacccttaggtatggtactgtatgcgtggaactaactttaagatatcctttctttgtagggcattgtgaaagaggtttgtttcttcagaaaaaagatttctttggatcttcatttttgagtcttacatatggcaatggggtttgtgtatgaaatgtaactcagagaacgttacctatttcattgatgttgcctaattgtgattggtggaaatgatattagaaaCTGTGTATGATGTAACTAAAAAATAGTAATAAAATTTTTAAGTGTGTTTATGTATATGTATACATGAGAGAGTCTGTATTGTTTTAAATTCAAAGAAGGGTCTAGCAAGATGGCTCAGCTGGTAAAGATGCTTGCCACCAAGCCTCAGGACCTGAGTTTGCCCCCCAGGACCCATATGGTGGTGTGCACACACATATACAAGTAAATGTGCTTA
This mutation is a 592 bp deletion beginning at Chromosome 1 positive strand position 10,047,149 bp and ending after 10,047,740 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 38734 | TTG TGG AGT TCT CCC CTT TG | Wild type Forward | A | |||
| 38735 | GGC TGT TGT TGG CTG CTT | Wild type Reverse | A | |||
| 38736 | CCC CAC TCT TTA CAT ATC ATG C | Mutant Forward | A | |||
| 38737 | ACA TCA TAC ACA GAC ACT AAA CAA ATG | Mutant Reverse | A | |||
| 38738 | Fluorophore-1 | TGG CTA AGG AGA ACA TTC C | Quencher-1 | WT Probe | ||
| 38739 | Fluorophore-2 | ACA TGC AAG TAC AAC ATC ACA TAA CTA A | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 38734 | 0.40 uM |
| 38735 | 0.40 uM |
| 38736 | 0.40 uM |
| 38737 | 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.