Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 125 bp
Wild Type = 169 bp
>chr8:32276899-32277067 169bp CTGACTGGTCTGTGCAATCG GGTAGCACTGCCCTGAAGAG
Wt Sequence (deletion in lower case):
AGTAGTTACTAACTATAAATGAACTTCTGTTGCTGAGTAATAGGGTATGGTCGTATCTGCATTTAGGTTTTTCTTGCGACTTTGTTGTTGTTTATAAGAATATATGTATTCATGTTAGGGGACGAGCGAGGCCTATTAAAAGATAGCTCTATCTTTTCATTCCCTAAAGACCCTTTCCTCACTGTGACACCCAGCAATGTGCTCTGACATGGTCCTCATCATCTTGTGTGTTTGTGCTTTGGTGGTGGTGTTGCTGgagcctacagtgagggcaaggattgagccatggagcaccagctgcaggccagagagcacggcctctttttatattaaacagcatctttggggccagagcaaaagggctttctttctgtcttgcaggtaatctggaatacactaggaaacaagcagttttgttctttcaaaataaggtgagtagattgcagcctagactaaaatgtgttcggccgcccgcttggttgtaaacatttctttccttgcatcctagaaccaagactcagggcttctgcagaaatgaatacagtctgactggtctgtgcaatcggtcgtcctgtcctctggcaaatagtcagtatgctaccattaaagaagagaaaggtacgggagctacacgacaatttataaacgGATGTCCCCAGCCTCTGTAAATAACAATAATAAAGCTGTCTAACTTCTCTTCAGGGCAGTGCTACCTGTACATGAAGGTTATAGAGCGAGCAGCTTTTCCCAGGCGTCTCTGGGAACGGGTAAGATTCAGTGAGAAAACTCTAGTGACCATTGGGCAAAAGCAAAATTGCCATGTAGGCCATCTCTGCACTTGAAAGCCACCCTCATAGATACCG
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
39027 | CTG ACT GGT CTG TGC AAT CG | Wild type Forward | A | |||
39028 | GGT AGC ACT GCC CTG AAG AG | Common | A | |||
39030 | Fluorophore-1 | TCC TGT CCT CTG GCA AAT AGT C | Quencher-1 | WT Probe | ||
40126 | TGT GCT CTG ACA TGG TCC TC | Mutant Forward | A | |||
40127 | Fluorophore-2 | CTT TGG TGG TGG TGT TGC TG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
39027 | 0.40 uM |
39028 | 0.40 uM |
40126 | 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 |