Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chrX:101214535-101214634 100bp GAAATGGAGGGAAGGAAGGA AATAGCAGCCCAGTTGGAGA
Mut = 102 bp
Wt = 100 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
ACAGGATTATTAAAGAAAGTGAAGAGTACTTGAAGTTTATTTCCTTCATTGAGATAGTAACTCTGTCCTTCCACAGAAATCAAGTTGGATTCATATGAGCCATGCTTACTGTTCTAAGGTCTTAGCTGGACCTGGGgcattatagatcccaagcagttcttttataagaactgagaaataccctttacttagcaggtggcggcagaaggatcagaagttcaagggcatccttggctacttagacagtttgagactaccctgcagccatggaaatggagggaaggaaggagggatggaggaagagggaggacacattttaccatctcatttgctttcttctttcttgccctctccaactgggctgctatttgactgcagatcgtagtaccaccactgcctgggaaagccttgaagcggcagctccctttccgaggagatgaagggatctttgaggagtctttcattgaagaaaggaggcaaggcctcgaacagtttattaacaagtaagccaaattcttggctgctttgcatactgcttttgccattatgaaatgtttctttaccaccctcccttccccatttttctctctcctccctgtcctcctTCATTCCATACTTGGTTCTTTCCCTTTTTGCCTGGGAATGACTCCACAGCCTTGCATGTGCTAGTCAAGCACTCTACCTCTAAACTATTATACTCCCAGCCCATCATGATGTTCTTATATATATTTTTGTCTACTTTG
This mutation is a 469 bp deletion beginning at Chromosome X position 101,214,299 bp and ending after 101,214,767 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
50451 | GAA ATG GAG GGA AGG AAG GA | Wild type Forward | A | |||
50452 | AAT AGC AGC CCA GTT GGA GA | Wild type Reverse | A | |||
50453 | CTC TGT CCT TCC ACA GAA ATC A | Mutant Forward | A | |||
50454 | AAG GGA AAG AAC CAA GTA TGG A | Mutant Reverse | A | |||
50455 | Fluorophore-1 | AGG AAG AGG GAG GAC ACA TTT TAC C | Quencher-1 | WT Probe | ||
50456 | Fluorophore-2 | AAG GTC TTA GCT GGA CCT GGG TC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
50451 | 0.40 uM |
50452 | 0.40 uM |
50453 | 0.40 uM |
50454 | 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 |