Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 112 bp
Wild Type = 109 bp
>chr9:65042263+65042371 109bp GCCATGTCTGGTGAGAACAG AAAGATCCAAAAGGGGCTTC
Wt Sequence: AACTTTGGCAGTCTTGTTTTTCTTACTCAGGTTTTTTGTTTTGTTTCATTGTTTTTTGTTTTTTGTTTTTTGTTTTTTTTTTTTTGTTTTGTTTTGTTTTGAGACAAGGTTTCATTTAGCCTAGGCTGGCTTCAAAATCCTGATTCTCCTGCCTCTACCTCCTGAGTTGTGGGGTTATAGGTGTTTGCTACCATGCCTAGCTTAAAACATTGTTTTTATTCAAGGAAATTTCcacggcatgaagcatgtgtataccctacttaataacatcattttggttcctgtggaatggaaaatattataatatgtagaagacattatgtatttacgactgttgtaataaggacaatattacaaaataataatgtattttttgtttttgtttttttccttatagccatgtctggtgagaacagagaaaatacactattttattctgaaatccctaaaaccatcaacagagcagcagtcttaatgctttcctggaagccccttttggatctttttcaggtaggaacaagtattcttagtcctatgatttggtgttatttcttcatttatttcagggcaaatgtttttCTAGGGTTTTATATTTTAAGTAGATGTGAATAAAAAGGAATTTAAAATGTGTAATTGTTTTTACATGAATGACTTTAAATGACTAATTTCCTGTATCTAAAATCATAGGATATTGTAATCCAATAATTTTGGTGTTTCATTTGTTTGAAATGTTTTGGTTTGTGTTTGTGAATGTTTTGCTGGCATGTCTGTCTGTGCACCATACACATGCCTGGTGTGCATAGGCATTAGAA
Mutant Sequence:TCAAAATCCTGATTCTCCTGCCTCTACCTCCTGAGTTGTGGGGTTATAGGTGTTTGCTACCATGCCTAGCTTAAAACATTGTTTTTATTCAAGGAAATTTccTAGGGTTTTATATTTTAAGTAGATGTGAATAAAAAGGAATTTAAAATGTGTAATTGTTTTTACATGAATGACTTTAAATGACTAATTTCCTGTATCTAAAATCATAGGATATTGTAATCC
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
46332 | GCC ATG TCT GGT GAG AAC AG | Wild type Forward | A | |||
46333 | AAA GAT CCA AAA GGG GCT TC | Wild type Reverse | A | |||
48449 | Fluorophore-1 | ATT CTG AAA TCC CTA AAA CCA TCA | Quencher-1 | WT Probe | ||
49159 | Fluorophore-2 | ACC ATG CCT AGC TTA AAA CAT TGT | Quencher-2 | MUT Probe | ||
49160 | TGT GGG GTT ATA GGT GTT TGC | Mutant Forward | A | |||
49161 | TCT ACT TAA AAT ATA AAA CCC TAG GAA | Mutant Reverse | A |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
46332 | 0.40 uM |
46333 | 0.40 uM |
49160 | 0.40 uM |
49161 | 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 |