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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
>chr18:75611651-75611765 115bp CAACTCCCTGGACATGCTG ATGGCAGGAGATCCCACAC
Mut= 107 bp
Wt= 115 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
CACCGTCTCAGTGAGGGCCTGCACTTGCCACAGTGTCCAGGCCTGCAGTCTCCACAGTGTCCTCCTGATGCTCCTGCTACAATATTCCTTTAGGTGGCTCCCTCCAGCCCCTAGCCATGCAGGCTAGCCAGCCTTAGGTGTCAGCGAGGGCCCcagtggccctctcaggcttggacctactgagcctctacctagttgtttccagctggccccataagtaatgccagcccctctctttgtctctttcagaatggcaacaaggacaactccctggacatgctgggcacagatatctgggccgccaatacctttgattccttcaggtaacttcctcctTTTTTGTCAGATGTCCAGGGCTGGTGTGGGATCTCCTGCCATGCATCAGGACTTATGGGGTGGATCTGGTATGGAAACAGATGATGAGGCTGGAGGGGAAGGAGAGACTTTAGCAGAGGCTCCAAGAAAGGAAGAGCCAAAGGGCAGAGGTCAAAGGGAAGATAG
This mutation is a 183 bp deletion beginning at Chromosome 18 position 75,611,693 bp and ending after 75,611,875 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 43374 | CAA CTC CCT GGA CAT GCT G | Wild type Forward | A | |||
| 43375 | ATG GCA GGA GAT CCC ACA C | Common | A | |||
| 43376 | TTT AGG TGG CTC CCT CCA G | Mutant Reverse | A | |||
| 43377 | Fluorophore-1 | AGA TAT CTG GGC CGC CA | Quencher-1 | WT Probe | ||
| 43378 | Fluorophore-2 | CAG CGA GGG CCC TTT T | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 43374 | 0.40 uM |
| 43375 | 0.40 uM |
| 43376 | 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.