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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
>chr19:8797183-8797275 93bp AGGTGAAGTGGTGGATGCTG GAACCCTCACCTTAGGAAATACC
Mut = 117 bp
Wt = 93 bp
Fam = Mut
Hex = Wt
Sequence
Wt Sequence (deletions in lower case):
CTCTGACTCGTTTGTGGTTTTTGAAAGACCTGTCTCAGCTTTGACCCTGGCAAGTCCTCCTGCCTTAGCCTACATGGTACTGAGAGAATAGGTGTATGTCAGCATATCTAGGCCTTCTTGGGCATTTACTGTACCATCATGAGAGCATCATTTAATATTTCATGTGCTAAGCTGGTCGGGGGTGGCGCATGCCTTTAATTCCAGCACTTgggaggcagaggcaggtggatttctgagttcaaggccagcctggtctaaagagtgagttccaggacagccagcgctacacagagaaactctgtctcgaaaaaacatatatatgtgtgtgtgtgtgtgtgtgtgtgtgtatatatatatatatatatttaggtgctaatctgtgtcctgtctcttcttgatcaatgaaccatctgaacagaggttctttcacacagagactcttattgctggtggcttttcagaggctcttcacatagctgtcttctttcatttttcatgcagatatggctttgtaattgctgtcaccaccatcgacaatattggtgctggtgtgatccagccaggccgaggttttgttctttatccagtgaaatacaaagctattgttttccggccctttaaaggtgaagtggtggatgctgtggtcactcaggtcaacaaggtaagagtattattcatagaaaggttatGGGGTATTTCCTAAGGTGAGGGTTCAGTCCCTAGTCCACTTACTAGTTGCCTTAAGTCTTTTGTTGTTGTTAAGATTCATTTTATCTTAGTTTTTTGGTTTTTCTTTTCTGGTTTTAGGGTTTTCTTTTTTCTTTTCTTTTCTTTTCTTTTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTGAGACAGGGTTTCTCTGTGTAGCCCTGGCTCTCCTGGAACTCACTCTGta
This mutation is a 480 bp deletion beginning at Chromosome 19 position 8,797,208 bp and ending after 8,797,687 bp (GRCm38/mm10). There is a single bp insertion A at the deletion site.
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 50762 | GAA CCC TCA CCT TAG GAA ATA CC | Common | A | |||
| 50763 | AGG TGA AGT GGT GGA TGC TG | Wild type Forward | A | |||
| 50764 | TGG GCA TTT ACT GTA CCA TCA | Mutant Forward | A | |||
| 50765 | Fluorophore-1 | TCA CTC AGG TCA ACA AGG TAA GAG T | Quencher-1 | WT Probe | ||
| 50766 | Fluorophore-2 | CAT GCC TTT AAT TCC AGC ACT TAG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| 50762 | 0.50 uM |
| 50763 | 0.50 uM |
| 50764 | 0.50 uM |
| Glycerol | 6.50 % |
| Dye | 1.00 X |
| Kapa 2G HS taq polym | 0.03 U/ul |
| 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.