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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= 129 bp
Wild Type = 131 bp
Sequence
Wt Sequence (deletions in lower case):
AATGGGTCCTTCCACCTGCCTGGGAGACACTGTAATCCCCACTTGCATTATTATTGTAATTCCTACTTGCATATGAAGAAATGGTAATTAGAGGTGGAAGTGGCTTGCTTTCTCCTCTTCTGCCCAACCCCCTTTGTCCCTGTAACTCATTCACCTGGTGGAGGGAGTGGGTTTTGTAATTCACTTTTGGAGCTTGTGGGGGACAATGAGACCCATTGAAGGGCTTTTGTCTTGTTTTTCACCAGAACCTAGCTAGCATCCTCTTCAATTCCAACTAGAATGCTGTGGCAGGCACttcgaagatttggtcaaaagctggtgcggagacgtgtgttggagctgggtatgggtgagacccgccttgccagatgcctgagcaccctcgacttagtggctctgggtgtgggcagcacattaggtgcaggtgtgtatgtcctggctggagaggtggccaaagataaagcaggaccatccattgtgatctgctttttggtggctgctctgtcctctgtgttggctggactgtgttatgctgaatttggtgcccgggtccccggctctggttctgcatatctctacagttatgtcaccgtaggtgaactctgggccttcaccaccggctggaatctcattctctcctacgtcattggtgagatagatgcttggggtaggatgggagggatggatgattacacaaatagcagagggattatggatccagaaatccgagtgcaatTTCAGTGAAGTCTAAATTGTCTTTATTTTTCCTAGTATCTCAATACTTCTAGATGTCTGTTTGTTTGTTTATTTATTTATTTATTTGGGGTAGGGGTATAGGGAGGGTTTGAAGGTGCAGTACAATTGTTCACCTCCGTCTGGTGTTCATTCATTAAGATGTTAGTTAGGGTCTTTAAAATTAATAACAGGAGCTGGGTCTAGAGTTGTACACCTATAATTCCAGCACTCAGAGGCTAAGGTAGGAGGAT
This mutation is a 441 bp deletion beginning at Chromosome X position 101,083,784 bp and ending after 101,084,224 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 48309 | CTT CAA TTC CAA CTA GAA TGC TG | Common | A | |||
| 48310 | CAC TAA GTC GAG GGT GCT CAG | Wild type Reverse | A | |||
| 48311 | CCC CTA CCC CAA ATA AAT AAA T | Mutant Reverse | A | |||
| 48312 | Fluorophore-1 | AGC TGG TGC GGA GAC GT | Quencher-1 | WT Probe | ||
| 48313 | Fluorophore-2 | AGG CAC TTC AGT GAA GTC TAA ATT GT | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 48309 | 0.40 uM |
| 48310 | 0.40 uM |
| 48311 | 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.