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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
>chr2:13224647-13224768 122bp CTCTGTACCTCAGAAACAAG AGGGGCTGAAAGCAGAAATG
Mut= 121 bp
Wt= 122 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
GAGTTCAATGATAACAAGGACAGCTCCACAGGTACGGGATCATTTAAGAACTTGAGTGTACCCTCCTTCATTGGGGTTAGCTGTCTTCGGCTGGCAGTTCAAAGCAACCACAAGGAAGAAAATAACTTCAGCTGTTATGCTTGTAGTGTGGAAGTGTGTGCTGTGTTCTATTCCCTGAGAGGCTGCTGACAGCATGGGTCTCATGTCTTCCTCATTTCTGCTTTCCGCCCCTCTGTACCTCAGAAACAAGTCCTGCCAGGTAGTTTGATCTAATCAGACAAAAGGAAacacttcctgagaggctgctgacagcatgggtctcatgtcttcctcatttctgctttcagcccctctgtaactcagaaacaagtcctgccaggtagtttgatctaatcagacaagaggaaacacttccgttccacctggtcaagaatttttagccatgaaagtaagaaaacccacaaaatgcatgtggacagcactggggtttgaccttacttgcgttcaatagggaagtgtgtggctagcatcctttgccccttgaattcacatgctgcatctccatccctatagctgtgccaccaaatgtagcggaactgaagaacttggaggtactaaacttcttcaacaatcagatcgaggaactgcctacccagatcagcagcctccagaaactcaaacacctgaaccttgggtgagtattggaggagcttgaggaaatctccgccaggtaatgagctgaagccaatgcatgctgtgggcaagcaaGGGGAAGCGCTTATATGATTATCACATGGGAGTAGTGGGTTTCTAATTGCTTTTAATGTGTCAGGTTTAAGAAAAGGCTTATTGTTTTAATATTTCCTTTCAGTCTTCTCCATTAGTGTATATCCAGTTAAAGCTAATTGAGAAGCTAATTGTTGGCACAACAAGGAATCTTTTCATGATATTTTGT
This mutation is a 481 bp deletion beginning at Chromosome 2 position 13,224,231 bp and ending after 13,224,711 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 46951 | CTC TGT ACC TCA GAA ACA AG | Common | A | |||
| 46952 | AGG GGC TGA AAG CAG AAA TG | Wild type Reverse | A | |||
| 46953 | CTG ACA CAT TAA AAG CAA TTA GAA ACC | Mutant Reverse | A | |||
| 46955 | Fluorophore-1 | ACT TCC TGA GAG GCT GCT GA | Quencher-1 | WT Probe | ||
| 46956 | Fluorophore-2 | CAA AAG GAA GGG GAA GCG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 46951 | 0.40 uM |
| 46952 | 0.40 uM |
| 46953 | 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.