Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 119 bp
Wild Type = 119 bp
Wt Sequence (deletions in lower case; bp changes in brackets with wt first and insertions with carrots ^g^ (g insertion): CTCCCAAGTGCTGGGATTAAAGGCGTGTGCTACGACCGCCCAGCGGGAAGATTTTAAATAAACTAGGTAAACCATCTAGCCCGTGTCtagtaagtgcttggtaacaaactgctgtgtaggataataaggaaatacactttcctgatcggctctaggaactttgtgaaggaagctgaggagatcagctccagccgccgcatgaacacgctgactctaaaccggcacacagaaatcctggagattttagagataccccagcttatggacacgtgtgtccgaaacagctatcatgaagaggcgctggagcttgccgcctatgtacggcgactggaaaggaagtactcctccatccccgttatccaggtagccaccttgcccaggGGCCTCAAGCTGATGATCTGATAGTTAATTCTGTGATTGTGACTACACTTACACGTAGAAACCTTTGCATAGTTCATATCTTTCTGCCAGCCAGGTAAGCTG
Mutation details
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
49597 | GCC CAG CGG GAA GAT TTT A | Common | A | |||
49598 | CCT AGA GCC GAT CAG GAA AG | Wild type Reverse | A | |||
49599 | TGC AAA GGT TTC TAC GTG TAA G | Mutant Reverse | A | |||
51514 | Fluorophore-1 | CCG TGT CGG CCT CAA GCT | Quencher-1 | MUT Probe | ||
51515 | Fluorophore-2 | AGC CCG TGT CTA GTA AGT GCT TGG | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
49597 | 0.40 uM |
49598 | 0.40 uM |
49599 | 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 |