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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
>chr5:120499165-120499262 98bp GGAACAAAACTAGGGCTGGA CTGTGGGCAGACTCAGGTGT
Mut = 111 bp
Wt = 98 bp
Fam = Mut
Hex = Wt
Sequence
Wt Sequence (deletions in lower case):
AGGTCGGGAGAGGAGGCTGGTTTAGTCTTTGTTCCTCTGCCCCAGCACTTCCTGTCTCAGGTATGTAGCCCAGCAGGCATGTAGGTACAGACATGCAGGCTAACATCTGTATGGGCAGCTGCTGCTCACCAGGTGCAGACAGGCAGGCCAACATCTGTATGGGTGGCGGCTGCTCACCAGGCTGTTTTCATGGTCTAAAACTAGAGACTAGGCAGGGATGGTGTCACACATCTGTGATCTCAGCACGTGGAAAGCTGAGGCAGGAGGATTGGAGTCCCAGGCCAGCCTGGGCTACAGAGTGAGACCATGTTTGAAAAAGGGACAGGAACAAAACTAGGGCTGGAGCAGttgcctgccacacgagggcctgaactccatgtgtagtgtaaggaataaaaagccacacctgagtctgcccacagccgtgggtctggctgtgtggctgtcttttcaggggtccctgctgctaactgactgtccttcttctcccctccaggtgggcctatctggacctgagcacaaatggcaggtacaatgacagcctgcaggcctatgcagctggtgtggtggaggcctctgtgtctgaggaggtaaggccagtgtgggagtccagggtcccagctgcctgcccctggccaagcccagacatcctgagggccgtcagtggggttgATGCATGTGTTGAGAAAGGGTCCCCATCTCCTGTCCTCCATTAGGGGAGAAAAAGAGCCCTGTTCTTCAAAGACAGCACATAGGCTGGACCTGGTGAAACACACCCACAATTGCATGACTTGGGAGGCTGAGACAGACTGCTGAGTTCAGGGGTAGCCTGGGTTAGAGACAAGAAACAACAAAAACTTCCAGGCTATGATTCATGCCTGTTGTCCTAGCACTGGGGGATGGAGGGAAGACAAGGGTCAGGAGATCAGAACCAGCTGTGACTACTCCCC
This mutation is a 324 bp deletion beginning at Chromosome 5 position 120,498,915 bp and ending after 120,499,238 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 49129 | GGA ACA AAA CTA GGG CTG GA | Common | A | |||
| 49130 | CTG TGG GCA GAC TCA GGT GT | Wild type Reverse | A | |||
| 49131 | CAG CCT ATG TGC TGT CTT TGA | Mutant Reverse | A | |||
| 49132 | Fluorophore-1 | CCA CAC GAG GGC CTG AAC T | Quencher-1 | WT Probe | ||
| 49133 | Fluorophore-2 | AGA TGC ATG TGT TGA GAA AGG GTC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 49129 | 0.40 uM |
| 49130 | 0.40 uM |
| 49131 | 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.