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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
>chr5:110652473-110652581 109bp GGCTCAGAAATCGTCTTTCC CTGACCAAGGTCCCAAACTC
Mut = 101 bp
Wt = 109 bp
Fam = Mut
Hex = Wt
Sequence
Wt Sequence (deletions in lower case):
ATAGACCATGCGATCTGGGAGGACGACGCAGTCACTCATTGTAGCTCAGGAAGCGAGTTGAGATCCGTATACTTGCCAGGACTGCGTGAATCCGTATACGTAGCCGTTTTCACTCACTCACACACACACACACACTCTCTCTCTCTCTCTCTCTCTCTCTCTCACACACACACACACACACACACACACACACACACACACACACACACACACACACCCATGTTTTATTTGCGCCATTTCCGAGTTTGATACTGTCTCTGTCCTAGTGGGGAGACCCAGGTTCTGAGGCAAAGCGAGAGGAACTACATTTCAATACCCCCTTCCCAGTTTGCAGGGCTCAGAAATCGTCTTTCCCAAGCCCCGGtctggcatcttaaatagtttacagctgtgaagatgagaaacagccggaagattggggctgagtttgggaccttggtcaggcttggcacacataaatatgagtgtgagttagctctcctaactctgtccccagggtcccagggagccacatacaaatacaaggtgtggataagacatcgataccacagctgctgtaaccgcctagaggaactcctggcccatcccaccttccaggtcaaggtgagtcttgaagcccttagcccagcttttcctctcatctctaaAGTACTTCACAGCACAGGGGTAGAGGCCTTTGATGCGAGCATCTCTTCCTTCACTTGTCTGAGAGACTTCTACCTAGACATCTTTATTGCTGTTCTTTTCCACTGTGTGCTGTGAATCATCCGGACCTAAAGACATGGAGTCTGTAACCCTAGTTCCAGGCAGCAGATCTGCTTTGTACATTGTCACATTTGTTGAGGCACCTCCAAAACTGGGGGGGACAAGGGAATAGTTAGGTGGCCTCCCCTGCAGGCCCACCCTGTGTACATGCCTCAAGCCAAGTTCTGTTAAGAGTTCTATCCCAGGTCACCATCGGCACACACATGTAACATGGA
This mutation is a 283 bp deletion beginning at Chromosome 5 position 110,652,269 bp and ending after 110,652,551 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 49260 | GGC TCA GAA ATC GTC TTT CC | Common | A | |||
| 49261 | CTG ACC AAG GTC CCA AAC TC | Wild type Reverse | A | Wt R | ||
| 49262 | AGA AGT CTC TCA GAC AAG TGA AGG | Mutant Reverse | A | Mut R | ||
| 49263 | Fluorophore-1 | AAC AGC CGG AAG ATT GGG G | Quencher-1 | WT Probe | ||
| 49264 | Fluorophore-2 | CCG GAG TAC TTC ACA GCA CAG G | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 49260 | 0.40 uM |
| 49261 | 0.40 uM |
| 49262 | 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.