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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr6:113025913-113026105 193bp CTGGCTTCTGAGGACCG AATCTGTGGGAAGTCTTGTCC
Mutant= 140 bp
Wild Type =193 bp
Wt Sequence:
AATCTGTGGGAAGTCTTGTCCCTCCAATTTTACACCTGTTCAATTCCCCT GCAGGACAACGCCCACACACCAGGTTAGCCTTTAAGCCTGCCCAGAAGAC TCCCGCCCATCTTCTAGAAAGACTGGAGTTGCAGATCACGAGGGAAGAGG GGGAAGGGATTCTCCCAGGCCCAGGGCGGTCCTCAGAAGCCAG
Mutant Sequence:
TTTGCTTCTGTGTCTTCAGCAAAAAGTTTTTCTACCAATTCCAACTTGAATTCcatggtggcgaattcggatccaagcttcgaggtcgacggtatcgataagctagcttgggctgcaggtcgagggacctaataacttcg
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
21306 | CTG GCT TCT GAG GAC CG | Wild type Forward | A | |||
21309 | AAT CTG TGG GAA GTC TTG TCC | Wild type Reverse | A | |||
21310 | Fluorophore-1 | TAA CCT GGT GTG TGG GCG TTG T | Quencher-1 | WT Probe | ||
45556 | TTT GCT TCT GTG TCT TCA GCA | Mutant Forward | A | |||
45557 | Fluorophore-2 | TGA ATT CCA TGG TGG CGA | Quencher-2 | MUT Probe | ||
oIMR7882 | CGA AGT TAT TAG GTC CCT CGA C | Mutant Reverse | A |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
21306 | 0.40 uM |
21309 | 0.40 uM |
45556 | 0.40 uM |
oIMR7882 | 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 |