Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr2:180902350-180902466 117bp ATACTTTTGTGGGGCGTAGC CATGCTCTATCAGCCACAGG
Mut= 157 bp
Wt= 117 bp
Fam=Mut
Hex=Wt
The wt reverse primer (primer 58900) anneals over the nucleotide sequence containing mouse genomic variation rs250950845. The mutant reverse primer (primer 58901) anneals over the nucleotide sequence containing mouse genomic variation rs253270638
Wt Sequence (deletions in lower case):
TAGCAAACCATAGAACACTGTTGAGTCAAGAATGTGGTTCTCCTGACTTTGAGGTGGACATGTGTAAGCACTATGCAAGACAGAGCACTGTGCATTTTACATTTCTGTGGCTTCTTAGTGGGCTCGTCCAGACAAGGTATCATCCATTGGCAGGCTATATGCTTGTGTTTTGATTAATATCATGTTTTTCTGATATGATATGGGTGAATATCACTAAAGGAATAAGTTATCCATAAGAAAGAGATTTGCTGTGTTCTTTCTAGTATAAGAGTACATAAGCTCTCTTCTCTGCTGTGGTTTGACCCTCATCTCCTACATAATATACTTTTGTGGGGCGTAGCATGCCAAGAATCCGGTAGAGaactggctactggggatattccaatagagcactatatcagagtgttctatactgagacctgtggctgatagagcatggttgtctttcttgctgtgaacctctagtgcaggctggagcaggaagggagaacaggctgtgctgcagtgtcaccaggcctgtgtcaggctgagaaggtcctctactgtcaccaggcccgtgtcaggctgagaaggtcctctactgtgtttctttctagtatgatgaacatgtgatcagcccaaaggagtttgtgcacctggcagggaaatctaccttgaaggattggaagagagccatccggatgaatggcatcatgctcaggtgggcatctgacgagtctgagatGATGGGAGGGGTTTGTAGCCATTGCTTTTTCATTTGATTTTAGATTTGTGAAGATGGAGCATTTTAGTTGTTTATTTTAATTTCCACTAATTCTTTACTTGGTGGTAGTGGTGATGGTGATGTGTTTATAATTTGAATATGTCACTGTTAAAACTTAAGGATATAATCTGGTGTTTGTGCATAGATAAACATTTAGGCTTACTTTTTTAACAGCTCTTTATGCTACCCTTTACTTCTGTCTTTCATTAAAGTCCCTTGCAAAGTGCACA
This is a 363 bp deletion beginning at Chromosome 2 position 180,902,064 bp and ending after 180,902,426 bp (GRCm39/mm39)
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
58899 | ATA CTT TTG TGG GGC GTA GC | Common | A | |||
58900 | CAT GCT CTA TCA GCC ACA GG | Wild type Reverse | A | |||
58901 | CCA TCA CCA CTA CCA CCA AGT | Mutant Reverse | A | |||
58902 | Fluorophore-1 | ATC CGG TAG AGA ACT GGC TAC TGG | Quencher-1 | WT Probe | ||
58903 | Fluorophore-2 | ATC CGG TAG AGG ATG GGA GGG GT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
58899 | 0.40 uM |
58900 | 0.40 uM |
58901 | 0.40 uM |
Wt Probe | 0.15 uM |
Mutant Probe | 0.15 uM |
DNA |
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 |