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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
>chr15:8082689-8082798 110bp CCTTTACTGTTGAGCCACCTG AGAGCTGGGTCTCATCCTGA
Mutant= 102 bp
Wild Type = 110 bp
Sequence
TGGAGCTGGTCTTAAAGGCTGTTGTGTGTTGCCTGCTTGCCTTTTGGGAACCAAACATGGGTCCTTTGTAAGAGGTCTCACTATGTGGCCCTCTGTCACCCAGGCTTGCCTCCACCATACAGAGATCTACCTGCCTCTTTCTCCCTAGCTTTGGGCTTAACCGTGTGTGTGCCACCACACCATCCTTTACTGTTGAGCCACCTGTGGCTCTGCCTCTGCTTCCCGggggattccattccagatgtgtgccacctctcctgattcttgctctgctcaggatgagacccagctctgtttgttttgggcaggccttctagtcacactgtctttgtaacttaattttcagcctaatattgataaatccgactgatggaaattgtgtttacttttaacttacagaagcaagagaaaaagaggaagaaatgaacagagaaaaagaattaagaaaacaaattgaagatatggagccagcaccgtcaagctcgagtgcagccagagagcgctcacagtcatcatgcaggtgcctgggcttccgcaaggcttctcaacgcccattctccatgtggccataatgcagtgtagtgagaaaagatgaactgtttctccaccgtagacagttgtgtacctaggacatggaaatgctgagtcagctttggagctggggatcttgtgatacagGGTTTTAAGAGAAAGTGATGTAGAAACACTTGTCCCATGCATTTTGTTGAAGTAGAAGCCATTCATTGTAAAGTTTGTTTTTGGCTCCTCTTGATGGCTACGTTGTATAGGCAGGCAGGCAGGCGTCCAACACTGTTCTAGGGATGTGCCAGGTTTGTGATTTTGATCATTTTCTTTCTTGTTTTAAATTTCTTTTTAATTTTTTATGTTTATTTATTTACATTTCAAATGTGATCCCCC
A 453 bp deletion beginning at Chromosome 15 position 8,082,304 bp and ending after 8,082,756 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 45247 | GGC TTC TAC TTC AAC AAA ATG C | Mutant Reverse | A | |||
| 45248 | CCT TTA CTG TTG AGC CAC CTG | Common | A | |||
| 45249 | AGA GCT GGG TCT CAT CCT GA | Wild type Reverse | A | |||
| 45250 | Fluorophore-1 | CCA GAT GTG TGC CAC CTC T | Quencher-1 | WT Probe | ||
| 45251 | Fluorophore-2 | CTT CCC GGG TTT TAA GAG AAA G | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 45247 | 0.40 uM |
| 45248 | 0.40 uM |
| 45249 | 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.