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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
>chr4:40164527+40164617 91bp TGAGGATTGGCTTTCAAGAGA GTAAGCGTCCTGGGGATTAAG
Mut= 96 bp
Wt= 91 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
CAGACATTCAAATTCTGAATGTCTGTTCACTGTACATAGTTTTCACCTTATAGACATTTTAAACTTGTCAATCACTTGGCCCTCTTTCCCTGTTAGAAGAATAATCCGATGTCATGTGTCAAGTTTATCTGAGGATTGGCTTTCAAGAGAGTTGTGAAAAGCAGGTGCCctgtggtttgaggttattaatgctttctaccttaatccccaggacgcttaccattttctatcagagttctcctggaggcagccgttcggaactgtgatgagtttctggtgaagaaaaatgacatcgagaatatcctgaattggaatgtcatgcaacataagaacatagaagtgccgtttaagccagcccgagtcatactgcaagactttacgtgagcctgacacctcacctcacctcacctctctctctctctctctctctctctctctctctctctctcgctcgctcgctcgctcgctcgctcgctcttccctgcccccccctgcctcccttgtcagcctcccaagaaagtttttactgtagcaaaaatgctttgtctctgagaaaccaggagataaaaggaaggcaaggggctcctggaacaagagaagcctgctctgtctctagtagtcaccccgcctcaccagtgacAGGGGTACCATCCTCAGGACTGATCACACTGGGAAGGGTAGAAGACAGCACGCAGGAGGGCTGTGTGGAGTCTCTTGTGCAGGCTGCTGTGCCTGCATACAGTTGTGCCTACCGACCTCAGGGATAGACGAGGTGCTTCGTGAGGCCTGGCTTGCACTCGGCTTGCA
This mutation is a 471 bp deletion beginning at Chromosome 4 position 40,164,567 bp and ending after 40,165,037 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 44894 | TGA GGA TTG GCT TTC AAG AGA | Common | A | |||
| 44895 | GTA AGC GTC CTG GGG ATT AAG | Wild type Reverse | A | |||
| 44896 | CCT GCG TGC TGT CTT CTA CC | Mutant Reverse | A | |||
| 44897 | Fluorophore-1 | CTG TGG TTT GAG GTT ATT AAT GCT TT | Quencher-1 | WT Probe | ||
| 44898 | Fluorophore-2 | AGG TGC CAG GGG TAC CAT | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 44894 | 0.40 uM |
| 44895 | 0.40 uM |
| 44896 | 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.