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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.
>chr11:32279355+32279447 93bp GTTGCTGGGTAACGAAGCTC AGGCACTCCAGGGACATAGA
Mutant= 120 bp
Wild Type = 93 bp
Wt Sequence: gttgctgggtaacgaagctcagtatcccacagagcctcctagaattcTTaaaagcccactaaaggtttacctttctatgtccctggagtgcct
Mutant Sequence: gttgctgggtaacgaagctcagtatcccacagagcctcctagaattctGCAGATATCCATCACACTGGCGGCCGCTGTCGACTCTAGAGGATCTTCTGCCAGACCCCCAGGAAATGACTT
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
35298 | Fluorophore-1 | CTT AAA AGC CCA CTA AAG GTT TAC C | Quencher-1 | WT Probe | ||
35299 | GTT GCT GGG TAA CGA AGC TC | Common | A | |||
35300 | AAG TCA TTT CCT GGG GGT CT | Mutant Reverse | A | |||
35301 | AGG CAC TCC AGG GAC ATA GA | Wild type Reverse | A | |||
35302 | Fluorophore-2 | GAT ATC CAT CAC ACT GGC GG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
35299 | 0.40 uM |
35300 | 0.40 uM |
35301 | 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 |