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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= 100 bp
Wild Type = 98 bp
>chrX:74152402-74152499 98bp GGGTTACTCACTGCCAGCAT TCACCTGTTTTTCCATCAGTG
Sequence
Wt Sequence (deletions in lower case):
TGGGTTGAGATTTAATTTGCATGTCATGAAACAATAAGCCCTACTAGCAGTCATTCCTTGTCACTCCTGGTTCCCAGTTCCTGGCACCCACTCATATGACTTGTTTGTGGATTGCATACAAATGGAACTTTACACAATGAGGTCTTTATAATAAATATTCATTTATGGAGTAGATACTGCATCTTCAACACTGCTCCTGGGAGCATTTGGGGGGATAGCTTAAAATTTAATTTCTATACTTTCTAGTCTATTGCACACTTGGTAAAATAGTAGAGAATAATTGTTAATAAGATCTATTAGTGAAAGTAGTGTAAGTATTCCCAGGCAGAGATAGAAGATCCAGCCCCCTTTGACAGAATCCTAGGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTATGTTTGTGTAGGGATGTTGTTAGTAAATCTAGCTTCGGGTTACTCACTGCCAGCATGAGCTCTGAGTAAAggtacagagggagtgttactttctctcttgcatcctagacaatcactgatggaaaaacaggtgaatgatcacttgcagagatacctggatgagatttgccacctcaaacagcatctggcatgcactgaagagaaaatggcctatttgtcctatgaaagagccaaggaaatatgggtgagggcactggggagaaaggactagtgagaaggatatggggatgaatagatgtatttggatatgcccaagccaagacaagagaagatatagagctgtgtctggcctgacagtacaatagtaacctggtttagagccagcagtgtccaccctgcatgcctgtgtgaactccactccttaccatgcagccaaggtgctttggagactttggacagtggttctaatcttgtagggtcaattactgactcataccctggaatctgtgtcctataaatcaggagtgaggcctgacaaacccaaggaatgctggggtgtttgcattgctagtagagagaaAGCCTTTGGGAATCTCTTCTTGGGGTCTAGCTTTTTGCCCCTCAGTCTTTTCCATACTCATAGCTCCTACTCCTGTTCCTGGTTAGACCTTTTTCACCTAGGTTTTCAGTCTCAGCAGTAGATAGTTCTCACCAGGACCCATCAGCCCAGCATTCCACAGCAATAGTCTGTCTCTGCTGGGCATACCCAAGAGGTTCTTCCCATCTTTTCTAAAAAATTCAACTCATCAGGTATGATGGCACATGCCTTTAATTTCAGGACTTGGGAGGCAGAGGCAGGCAGATCTCTGAGTTTGAGAACAGCCTGGTCTATCTAACAAGTTTTGGCTCACCTGGAACT
This mutation is a 510 bp deletion beginning at Chromosome X position 74,151,956 bp and ending after 74,152,465 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 44441 | GGG TTA CTC ACT GCC AGC AT | Common | A | |||
| 44442 | TCA CCT GTT TTT CCA TCA GTG | Wild type Reverse | A | |||
| 44443 | GAG CTA TGA GTA TGG AAA AGA CTG A | Mutant Reverse | A | |||
| 44444 | Fluorophore-1 | AGG TAC AGA GGG AGT GTT ACT TTC TC | Quencher-1 | WT Probe | ||
| 44445 | Fluorophore-2 | AGC CTT TGG GAA TCT CTT CTT G | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 44441 | 0.40 uM |
| 44442 | 0.40 uM |
| 44443 | 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.