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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
>chr7:140883371+140883455 85bp GGCGCTTGCTTTAACTCCT AAGGATGCCTTCAGCCTTG
Mut= 89 bp
Wt= 85 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
TAGGTTGTGGCATTGGAATTCCAGTTGACACTCAGAGTGGGAGTTGACGCTGTCTCAAAAAGTGTTCTCCAGTGTTACCTAAGAAAACTGAGTCTTGAAAGGATAAGTGACTAGGGGGTGCAAGCAAAGTCAGTCCAGAAATAGGCTTGAATTGGCTGGCGCTTGCTTTAACTCCTGGCTgtgcagtgggacctgggaaatacttggtgtttgatgggaccgtcaaggctgaaggcatccttttcttcacattaggatgtgggactgagggctttttgttttgtaggttgtggcatcagctgacacttgaggtgcttgattttgtgcaggacccatgctttgcccaaggagatggtctcattaaggtaatgacttaccagacagctcaGACGATGTTGAATATAATTACAAACTATTATTATTCATTTTTATTTACTTTTAACGTGTGTGCGCCCAAGGATGCCAGAAGAGATCTCCTGGAACTGGAGTTGCAGGCAGTTGTAAGCCAACTGGTGTAGGTGCTAGGAACCCAGTTTG
This mutation is a 208 bp deletion beginning at Chromosome 7 position 140,883,394 bp and ending after 140,883,601 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 41206 | GGC GCT TGC TTT AAC TCC T | Common | A | |||
| 41207 | AAG GAT GCC TTC AGC CTT G | Wild type Reverse | A | |||
| 41208 | GGC GCA CAC ACG TTA AAA G | Mutant Reverse | A | |||
| 41209 | Fluorophore-1 | CAG TGG GAC CTG GGA AAT ACT | Quencher-1 | WT Probe | ||
| 41210 | Fluorophore-2 | TGG CTG ACG ATG TTG AAT ATA ATT A | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 41206 | 0.40 uM |
| 41207 | 0.40 uM |
| 41208 | 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.