Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr17:30060842+30060967 126bp GCTTTGCTGGTAAGTGCAGA TGCAAAATCCAAAGGAGAGC
Mutant= 98 bp
Wild Type = 126 bp
Wt Sequence (deletions in lower case):
TACTGTTCCCAGTTGTAAACCGCAGCAAATAAAACAAGACATTGGCTCATGCAAAGGTAAAAGAAAACCCATGCAGCTTACTTTAGTTTTGTTTGCTTTTGTTTTGTTGGGGGTGGGATAGACAGACAGCTGGCCAGACTTGTTCTTAGCTTATTAGTTCAgttgtctggccagcaaattctttgcttgtatttaattttttgttttgattagaatgatacagaattttttagctcttctttctttcttttctttcttttcttttttctttcttttttcttttcttttcttttcttttttttttttttgccccaggtccagcaagactatgaatctctgttccaaatgctttgctggtaagtgcagaaaagggtgtgggtgttttctgtgtccccccccccccaacgatttatttattttttctttttggttattttaagattaaattatttagctctcctttggattttgcatGGGGCTTTGTTATGTTTGTGCGTGAAAGCAGATTCTCTTCACTGGGCGCTTCATATATTTGCTTAAGATTGCTAAACTCATCTCTGAGGGAAATGAAGTTTCATGTGTACTTGCATCAAATCCAATTTGCTAAGCTCTTGGGGTACAGAGGAATTATAAAAAACATGTCTGGTTTAGTAGGGTCCTGTTTATT
A 313 bp deletion beginning at Chromosome 17 position 30,060,656 bp and ending after 30,060,968 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
49692 | GCT TTG CTG GTA AGT GCA GA | Wild type Forward | A | |||
49693 | TGC AAA ATC CAA AGG AGA GC | Wild type Reverse | A | |||
49694 | GGG TGG GAT AGA CAG ACA GC | Mutant Forward | A | |||
49695 | GCC CAG TGA AGA GAA TCT GC | Mutant Reverse | A | |||
49696 | Fluorophore-1 | CTT ATT AGT TCA GGG GCT TTG TTA TGT | Quencher-1 | MUT Probe | ||
49697 | Fluorophore-2 | TGT GGG TGT TTT CTG TGT CC | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
49692 | 0.40 uM |
49693 | 0.40 uM |
49694 | 0.40 uM |
49695 | 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 |