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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
>chr11:28433784-28433921 138bp TTTCTGAGTGGTATACTGAACAGC TTTAATGAGTCCATTCCCATTA
Mut= 114 bp
Wt= 138 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
ACATTTCTGACAAAGCCTCAGCATCCTCATTTACAAATTTCTTTTATATTGTTAACATTGTGCAATGCTCAAAACAGTATAGTGTGAATGGCTTCTCTGGAAAGAATTGTCTTTCATTTTTAAAGAACTGACTAATCTCTTGGTCCTACTGAATAAATCAAGGCGAAGATATCCTTGACAGTAAACAAGACAAGACCTTgtggataatgtaagttctttctttttaattttagaatcaaccttgtcctatgttaggcagctggaggccagagtcagacagctggaagaagaaaatcgcatgctgccccaggtgggtgacttccagaagctcatacttagccagaatcatttgagttgtaaacttttatgtgaaatttaaagtctcaattctatgttctcctagaaataatgttgtaaagagtcatttatcatataaactggtgaggttccacgatggccattatcctaatttctgagtggtatactgaacagcaatcaataagaatgtgatgtgatttataatgaagaagagaataatatccattgtcctgcccatcatatcatGAGTGTGAGCTGGGGGATGGGTAATGGGAATGGACTCATTAAATTTGAAATTTTGATAATTTATTAATTCTGTAAGGTTGTAGGAGTTCATACTTTAGTTAAATTAGTGCTTTCTATAAGTCTTTGATAAAATTGAACTCTTTTAGTACAACAAAATAATGACAATAAGTGCCAGGTTTCACTTTGG
This mutation is a 365 bp deletion beginning at Chromosome 11 position 28,433,827 bp and ending after 28,434,191 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 46882 | TTT CTG AGT GGT ATA CTG AAC AGC | Wild type Forward | A | |||
| 46883 | TTT AAT GAG TCC ATT CCC ATT A | Common | A | |||
| 46884 | TGA CTA ATC TCT TGG TCC TAC TGA | Mutant Forward | A | |||
| 46885 | Fluorophore-1 | AGA ATA ATA TCC ATT GTC CTG CCC | Quencher-1 | WT Probe | ||
| 46886 | Fluorophore-2 | CAG TAA ACA AGA CAA GAC CTT GAG TG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 46882 | 0.40 uM |
| 46883 | 0.40 uM |
| 46884 | 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.