Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr5:129503672+129503759 88bp ATTGAATGGTTGACCAAAGC AGAGCTAAAGGCAGACAATCC
Mut= 84 bp
Wt= 88 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TGAGAGTTCGGGACTAGCAGGAAGAAGCGAGGAAATTTCGCCCGTTTGGTTCTGTCGGATAGGTATTTATGGACTCGGTCTGTGTGAATGTAAGCTGTAATATTTAACTACTGAGAACAGATATTTTTTTTGCAAGTGGCATTGAATGGTTGACCAAAGCATACATGGTAAGAACTGCTAGcaagggggtacgttttctgctctaaaggattgtctgcctttagctctaagttctgttgtgctgtagagaggtgcttagattttgttatgattactccagtttaaaagtaggagagagatgactaggcatattaagtataaatctgaagctttgcatttgttttaatatttttatttcattagtttaattggtaagtatgtgcatagagtgtcacacacaaccagcatgggtgtagacttccctccagtgaccagcctgttcacattacagatacgacgggcgtggtcacctgcatgacctctctgcgtacgatgctgagtacgccacgtggaaccgagactaccagctgtctgaagaggaggctcgggtagaggcgctgtgtgatgaagagaggtatttagccttgcatacggacttgcttgaggaggaggcaaggcaaggtactgctcaagaaagacttacttcagccacacctttttaaaattttaagtatttaaaaagttactcccgttcatttttatactcactctttctgatattatcttgacagtaccCAGTGGATTGGAAAAACAGGAGTCTTTGCGTTCTGAGAGGACCTCAGGATAGTTTATATGTAGAGCCACAGAGAATTTTCCCAGCTTTTGAGGGCAGACTGGGATTTGAAAAAACACAAAAACAAAAAACCAAAAAACAAAACTCTTTAACTGTTCTTCTTTAACAGTGTT
This mutation is a 554 bp deletion beginning at Chromosome 5 position 129,503,713 bp and ending after 129,504,266 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44152 | ATT GAA TGG TTG ACC AAA GC | Common | A | |||
44153 | AGA GCT AAA GGC AGA CAA TCC | Wild type Reverse | A | |||
44154 | GGT CCT CTC AGA ACG CAA AG | Mutant Reverse | A | |||
44155 | Fluorophore-1 | AGG GGG TAC GTT TTC TGC TC | Quencher-1 | WT Probe | ||
44156 | Fluorophore-2 | CTG CTA GCA GTG GAT TGG AAA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
44152 | 0.40 uM |
44153 | 0.40 uM |
44154 | 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 |