Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr2:154592040-154592157 118bp CTTTCCGGAGATGGATGG GCAAATTCAGGGACAAATGA
Mut = 99 bp
Wt = 118 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
GGTGAGGAAGGGGAAGGGCAGGTACTCACTAACTCGAGGTGTCAGGGAGTGACTCCAGTCTGCAGAGAGAGCTATCTATGCCCTGAGGACCTGGAGCACCCTGGCCTAGTTCAGATCTGTGTGTACATCCGAGaaacctgcctggaccagctgggcctgcactcatgggggctttctgtttgtgcagtctcagagagaagcttcaggccattctgaaagccacgtacatccactctcggaacctagcctgctttgtgtttgcctataagagtctccatgccctacagtcccatgtgcaaggcgagacccaccagatgcactctttcctggctgccttcatcggggggctcctgctgtttggagagaacaataacattaatagccaggtaaaggctttccggagatggatggggccagtggggtatcttttggttggcatctgtgtttaaggtcttaagaatctccagttcccATGTGGCCTTGTGTCTGTGTCATTTGTCCCTGAATTTGCCCACGGCGAGGGGGGGGGGTGTTTTTTGGGACAGGGTTTCCCTGTATACCCTTGTACACCTTACTCTGTAGACCAGGCTGGCCTTGAAATCGTAGAGACCTACTTGCCTTTTTCTCCTGTGTTCTGGCTTTAAAAGCATG
This mutation is a 339 bp deletion beginning at Chromosome 2 position 154,592,079 bp and ending after 154,592,417 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
51133 | GCA AAT TCA GGG ACA AAT GA | Common | A | |||
51134 | CTT TCC GGA GAT GGA TGG | Wild type Forward | A | |||
51135 | ATC TAT GCC CTG AGG ACC TG | Mutant Forward | A | |||
51136 | Fluorophore-1 | CAG TGG GGT ATC TTT TGG TTG G | Quencher-1 | WT Probe | ||
51137 | Fluorophore-2 | ATC TGT GTG TAC ATC CGA GAT GTG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
51133 | 0.40 uM |
51134 | 0.40 uM |
51135 | 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 |