Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 73 bp
Wild Type = 78 bp
>chr8:102636870-102636947 78bp CTGCATCGTCATTCTTCTGG TGGATAAATCTCTTGAAACGTGTG
Wt Sequence: GACGTGGAGGGTTCAGTCGGCAGAAGCAGGACTTCTACCTCCTGCCCATTGTGATCAGTGATGGTGGCATTCCACCTATGAGTAGCACCAATACCCTCACTATCAAAGTCTGTGGCTGTGATGTGAATGGGGCACTGTTGTCCTGTAACGCTGAAGCCTACATCCTGAATGCCGGTCTGAGCACTGGGGCACTGATCGCCATCCTTGCCTGCATCGTCATTCTTCTGGgtaaggatctccctgcccCATGGtgcaaccaatacacacgtttcaagagatttatccagtaaggctggctgcaaggttgtactgtgtccaatcacatggtttaaaacaaactgaaagatgtgatagctctcatgaagtctgttgcacaataggtagggcggctgagctggcaaattatggtggaagagtttgaggaaggccatagtgtgtactttatatggtatgg
Mutant Sequence: cccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatggcttctgaggcggaaagaaccagctggggctcgaccgCATGGTGCAACCAATACACACGTTTCAAGAGATTTATCCAGTAAGGCTGGCTGCAAGGTTGTACTGTGTCCAATCACATGGTTTAAAACAAACTGAAAGATGTGATAGCTCTCATGAAGTCTGTTGCACA
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
46681 | CTG CAT CGT CAT TCT TCT GG | Wild type Forward | A | |||
46682 | TGG ATA AAT CTC TTG AAA CGT GTG | Common | A | |||
46683 | CTG AGG CGG AAA GAA CCA G | Mutant Forward | A | |||
46684 | Fluorophore-1 | CCT GCC CCA TGG TGC AAC | Quencher-1 | WT Probe | ||
46687 | Fluorophore-2 | CTC GAC CGC ATG GTG CAA C | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
46681 | 0.40 uM |
46682 | 0.40 uM |
46683 | 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 |