Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 91 bp
Wild Type = 104 bp
>chr10:4746842+4746945 104bp TGCCCTAAAAACAGCAGAGA TCAACCAAGGAGAACAGAGAGAC
Wt Sequence: aatagtgtattgtctaaaaactgctctgtttatgccctaaaaacagcagagaacagaaaggaagacaaggtctttgtacagCGctatttgcacaacacatgaatatctagtaagtctctctgttctccttggttgaaacttggaaaagacacagactcccAGGagaagtggtaatgccagggaagctgtggctaagaatttctgcttccccct
Mutant Sequence: TAGTGTATTGTCTAAAAACTGCTCTGTTTATGCCCTAAAAACAGCAGAGAACAGAAAGGAAGACAAGGTCTTTGTACAGCcgcgccgattggccagtactagtgaacctcttcgagggacctaATAACTTCGTATAGCATACATTATACGAAGTTatattaagggttattgaatatgatcggaattatcagatctctagatccgcggtcgacatatgagcgctaagcttgacgtcaccggttctagaacctagGCTATTTGCACAACACATGAATATCTAGTAAGTCTCT
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
37084 | TGC CCT AAA AAC AGC AGA GA | Common | A | |||
47074 | GGT CCC TCG AAG AGG TTC A | Mutant Reverse | A | |||
47075 | TCA ACC AAG GAG AAC AGA GAG AC | Wild type Reverse | A | |||
47076 | Fluorophore-1 | CTT TGT ACA GCC GCG CCG A | Quencher-1 | MUT Probe | ||
47081 | Fluorophore-2 | CTT TGT ACA GCG CTA TTT GCA CAA C | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
37084 | 0.40 uM |
47074 | 0.40 uM |
47075 | 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 |