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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= 96 bp
Wild Type = 125bp
>chr5:100480108-100480232 125bp ACAGACAACTCAAGCATCTC TGCTAAAACATTTTAAAGTGACC
Sequence
Wt Sequence (deletion in lower case):
AAAACTCCTTCTTTTTATTATTGAATAGTCTAGTTGGAAAACTAATATTGTGTGTCGACTCTCATTATTAAACATTATTACTATTAAACTTTACTGTTTTAACCATCGgtgtgtttcagagagactagggatttattggaaagtagaggtgtaaggacacgtgcgcttatacataatgatcatattaatattttggtgagtggatttattttaaatgctttttactttaatatatagatatacttgctaaatgacctgactttactgtactactgtgtatgaatgaatgctgtctcattttactgcacacatgttttgtggaaacatatcttcatggttatttatgtttcagattgctaagaagcctcggacaccaacctcgggtccagtgatcacgaagctgatttttgcaaaaccaattaatagcaaagcagttacaggacagacaactcaagcatctccacccgtcgtcacaggttatctgtccttatagtttgtcagctttgtgtgtgtgtgtctgtcctagaTTTAGAAAAGGTTCTCGGTCACTTTAAAATGTTTTAGCATGGGCTAGAGAGATGGCTCAGTGGTTAAGAGCACTGACTGTTCTTCCGAAGGTCCTGAGTTCAATTCCCAGCAACCACATGGTG
Mutation details
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 70021 | ACA GAC AAC TCA AGC ATC TC | Wild type Forward | A | |||
| 70022 | TGC TAA AAC ATT TTA AAG TGA CC | Common | A | |||
| 70023 | GTG TCG ACT CTC ATT ATT AAA CA | Mutant Forward | A | |||
| 70024 | Fluorophore-1 | TGT CCT TAT AGT TTG TCA GC | Quencher-1 | WT Probe | ||
| 70025 | Fluorophore-2 | TGT TTT AAC CAT CGT TTA GAA AAG GT | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 70021 | 0.40 uM |
| 70022 | 0.40 uM |
| 70023 | 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.