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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 88 bp
Wild Type = 85 bp
chr8:85263616-85263700 85bp GCTGTAGTTTGTGCCTCTTCAC TCCAAACCCCATTCTATGAGAT
Wt Sequence: agttgagaagtattagctcaagaaagttggactagggttcttttggctgtagtttgtgcctcttcactgaaaggtacaccgtgcactAGTGTGAATGGCACTTGGaccatctcatagaatggggtttggaagaactttagaaagtgttcctattgatggttggttcatgaatattctttgttctatgtataccagGCATTTTCTCTATATGAAGATG
Mutant Sequence: AGCTCAAGAAAGTTGGACTAGGGTTCTTTTGGCTGTAgtttgtgcctcttcactgaaaggtacaccgtgcacttaattaagccaagcaggcataacttcgtatagcatacattatacgaagTTATTAGTGTGAATGGCACTTGGACCATCTCATAGAATGGGGTTTGGAAGAACTTTAGAAAGTGTTCCTATTGATGGTTGGTTCATGAATATTCTTTGTTCTATGTATACCAGGCATTTTCTCTATATGAAGATG
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
53273 | GCT GTA GTT TGT GCC TCT TCA C | Wild type Forward | A | |||
53274 | TCC AAA CCC CAT TCT ATG AGA T | Wild type Reverse | A | |||
53278 | Fluorophore-1 | CCG TGC ACT AGT GTG AAT GGC | Quencher-1 | WT Probe | ||
54103 | CCA AGC AGG CAT AAC TTC GT | Mutant Forward | A | |||
54104 | TCC AAA CCC CAT TCT ATG AG | Mutant Reverse | A | |||
55045 | Fluorophore-2 | TAT ACG AAG TTA TTA GTG TGA ATG GC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
53273 | 0.40 uM |
53274 | 0.40 uM |
54103 | 0.40 uM |
54104 | 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 |