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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= 109 bp
Wild Type = 100 bp
Wt Sequence: tggagacaaatgtagagacaaggggaaaggatgaacaagagttgagtcttaggagttctagATgtaggtgtccctcctgatgtcgcctctggacttccta
Mutant Sequence: TGGAGACAAATGTAGAGACAAGGGGAAAGGATGAACAAGAGTTGAGTCTTAGGAGTTCTAGAgtcagcttctgatggaattagaacttg
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
32955 | TGG AGA CAA ATG TAG AGA CAA GG | Common | A | |||
32956 | TAG GAA GTC CAG AGG CGA CA | Wild type Reverse | A | |||
32958 | Fluorophore-1 | TTC TAG ATG TAG GTG TCC CTC CTG | Quencher-1 | |||
33299 | Fluorophore-2 | AGT TCT AGA GTC AGC TTC TGA TGG A | Quencher-2 | |||
9715 | TTT GCC AAG TTC TAA TTC CAT C | Mutant | A |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
32955 | 0.40 uM |
32956 | 0.40 uM |
9715 | 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 |