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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
>chrX:10579434+10579539 106bp CACCATCGTGGTTTTTGG AAGAAAGCACATCACATCATGC
Mut= 114 bp
Wt= 106 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
GGATGTAGGCTCTCAGCTTCTGCTACAGTACTGTACCTGCTTCCATGCTCCCCACCTTGATGGACATGTACTCCCTCTGGAACTGCAAGCTCCCAATAAACTCTTTTGTTATAAGTTGCCTTGGCCATGGTCTCTCGTCACAGCAATGGAGGAGGAACTAAGACAATGTCTTTTGGCAATAGCTTCATGTTTCATGTTCTCTTCTCTCCTTTCGCAGATCActggggtgatcctgttggccgttggagtctggggaaagctgactttgggaacctatatctccctgattgctgagaactccacaaatgctccctatgtgctcattggaaccggcaccaccatcgtggtttttggcctctttggatgctttgctacatgccgtggtagtccatggatgctgaaaCTGGTGAGTACAACTCAGCATGATGTGATGTGCTTTCTTGGTTATTTTTATAAAACAATAAATTCATGCAGCAGTTAAGTAGCCCTTCTTGAAGCCACATACACACACACACACACACACACACACACACACACAAAAGACTTTAATATTCATTGATTTTAATTTTACACTAGACTCTTAACTAAT
This mutation is in a 182 bp deletion beginning at Chromosome X position 10,579,319 bp and ending after 10,579,500 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 52423 | CAC CAT CGT GGT TTT TGG | Wild type Forward | A | |||
| 52424 | AAG AAA GCA CAT CAC ATC ATG C | Common | A | |||
| 52425 | TGG AGG AGG AAC TAA GAC AAT G | Mutant Forward | A | |||
| 52426 | Fluorophore-1 | ACA TGC CGT GGT AGT CCA TG | Quencher-1 | WT Probe | ||
| 52427 | Fluorophore-2 | CCT TTC GCA GAT CAC TGG TGA | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 52423 | 0.40 uM |
| 52424 | 0.40 uM |
| 52425 | 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.