Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination. The transgene genotype is determined by comparing ΔCt values of each unknown sample against known homozygous and hemizygous controls, using appropriate endogenous references.
Mutant= 159 bp
Wild Type = 190 bp
>chrX:153596983-153597172 190bp TCCTCAAGCTTATTTAGTGCCTTAC TGTCCTTCCATGCCCTGA
Wt Sequence: atgccaagagtcgaataaacatttcttgggtgcaaaggtgttgtgaatggaaaatattacaaagcccagatctagaacatattcctatttaatatttaaggaaattggaataatccttatttcttggtatccaaagctgtctgaaactcctttccatactaaggtaaagcttagatatatacccaatgggaATATATATATATATATATATATATATATATATATATATATATATccaatgggaatatatatcagaatatgagttctgagtctaggtaataaaagccattatgacttccaacttgcttttcttgatttatcactctgggagaagccaatcaccatgtcattgggccacttaagcagattgtggataggcccacacgagaggctccagagggcttctgcccatttctagtaccatgcagatcctcaagtccaagcatttgggtcagtacaacgaattccttcattttgactgcagtcttccaagagaccttcagacagaccattcccccaatagctgcttccaaattcctggaccagagaaaatgaaaaa
Mutant Sequence: ATGGAAAATATTACAAAGCCCAGATCTAGAACATATTCCTATTTAATATTTAAGGAAATTGGAATAATCCTTATTTCTTGGTATCCAAAGCTGTCTGAAACTCCTTTCCATACTAAGGTAAAGCTTAGATATATACCCAATGGGAatatatatatatatatatatatatatatatatatatatatatatGTATATGGTGTGTGTGTCCAGTATTATTAGCatAAGAGCCATAGCTACATTGAAGAAACTGAGGTTCAGAGCTATAAATTATTGTAGGT
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
35058 | GCT GTC TGA AAC TCC TTT CCA | Mutant Forward | A | |||
35059 | GTT TCT TCA ATG TAG CTA TGG CTC T | Mutant Reverse | A | |||
35060 | TCC TCA AGC TTA TTT AGT GCC TTA C | Mutant Forward | A | |||
35062 | Fluorophore-1 | TGG TGT GTG TGT CCA GTA TTA TTA GC | Quencher-1 | MUT Probe | ||
35063 | Fluorophore-2 | AGG AAC TAA GGC TCA GCA GAC AG | Quencher-2 | MUT Probe | ||
40367 | TGT CCT TCC ATG CCC TGA | Wild type Reverse | A |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
35058 | 0.40 uM |
35059 | 0.40 uM |
35060 | 0.40 uM |
40367 | 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 |