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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
>chr16:20114348+20114479 132bp AAATCCTTGTAGATATTTTCTGTGG AAGCCCTAACGCCATGACA
Mut= 105 bp
Wt= 132 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
TTCCAGGGTATCCAGTATCCAGTCCCATTCTTTGACCTCCGTAGAAACCAAGCTCATGGGTGGTACACATACATACATGTAGGCAAAACACTCATACATGTAAACCTAAAACACTTTAAAAGAAAAATCCTTGTAGATATTTTCTGTGGCATAATGATGCCACTAATGAATCATTGCAAAGTAAATCACTGGCAGcctgtactgttcagcatatctgcaaggacaaatttaattgcctgtcatggcgttagggcttgattatattttaacagtcttgattatattttaatataccacttaactattatttgtgcattcttaacaataactattttaggtctactggctattcagaggtgatagttgtggttggaggctgtgaacgagttggaggatttaatcttccatacactgagtgctatgatccagtaacaggagaatggaagtccttggccaagcttccagaatttaccaaatccgagtatgcagtctgtgcactgaggaatgatattcttgtttcaggtaaatacaaattattatagtagctacaacttttgattgggaatcagattggctgtttttaaatgataatgacttactgagtgtaaaatgaatatttcagataaacaagtttatacaacaaaactgaaaagactttttgcccttgaaagcAAGAATACTTACAAAAGTTCTACCAAAAATTGGTTTGGTCATAATATTTTAAATGCTTTTATGTACATTTAGAGTATATGTATTTTAACTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTTTATATATGTGTGTGCCCAGAAGAACATAGAAGTG
This mutation is a 476 bp deletion beginning at Chromosome 16 position 20,114,420 bp and ending after 20,114,895 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 44142 | AAA TCC TTG TAG ATA TTT TCT GTG G | Common | A | |||
| 44143 | AAG CCC TAA CGC CAT GAC A | Wild type Reverse | A | |||
| 44144 | CCA ATT TTT GGT AGA ACT TTT GTA AG | Mutant Reverse | A | |||
| 44145 | Fluorophore-1 | CTG TTC AGC ATA TCT GCA AGG A | Quencher-1 | WT Probe | ||
| 44146 | Fluorophore-2 | AAA GTA AAT CAC TGG CAG CAA GAA TA | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 44142 | 0.40 uM |
| 44143 | 0.40 uM |
| 44144 | 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.