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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
Mutant= 119 bp
Wild Type = 131 bp
>chr11:99486755-99486885 131bp TGTCTGTCCTGAAAGTCACTGG GGGCATTGGCCATCTTAC
Sequence
Wt Sequence (deleted region in lower case):
GTCTGATTTCCAGTGGCTCTTGAACATATATGGTGCGCTAAAGTCATGCATGCAGACACACACACACACACACACACACACACACACACACACACACAAAACCTTTTTGTAAAAGGAGAATAGTTTGTCTGTCCTGAAAGTCACTGGTGGAAGAAACCTGGATGCAGACACactgctgtcatggctggatctgtccaactggaataattccgtgaacatattttacagatagtggacggtaagatggccaatgcccacattgtcgtgctcattgacaacgccaggatggcagtggatgacttcaacctgaagtaagtcccttcctcacagagttccacagaggcaaagctcggcctgccataacctgctgccaaaatcttgctggattataactaaagggcgggacaaaagtcacatgcaatttaagtaccccaggtacacgtgcacGTGATAGCATGTGAAGATTTTAATACAAACCAAGAATTGGGTCGTATTAAGAACTTTCCACTTCAGGTACAGTAATATTGTCATACTTACTTAGAAAAAAAATTTTTTTCCTTAAAGAAAATGTACTT
276 bp deletion beginning at Chromosome 11 positive strand position 99,486,564 bp and ending after 99,486,839 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 37241 | TGT CTG TCC TGA AAG TCA CTG G | Common | A | |||
| 37242 | ACT GTA CCT GAA GTG GAA AGT TCT | Mutant Reverse | A | |||
| 37243 | GGG CAT TGG CCA TCT TAC | Wild type Reverse | A | |||
| 37244 | Fluorophore-1 | CAA ACC AAG AAT TGG GTC GT | Quencher-1 | MUT Probe | ||
| 37245 | Fluorophore-2 | CTG TCA TGG CTG GAT CTG TC | Quencher-2 | WT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 37241 | 0.40 uM |
| 37242 | 0.40 uM |
| 37243 | 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.