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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.
>chr5:125963064+125963186 123bp AGCTCCTCCCAGACATTCAG GCAAAACAGCAACTCTTCAGC
Mutant= 108 bp
Wild Type= 123 bp
Wt Sequence: agctcctcccagacattcagggccatcagagtcaccgagtccatggcacgtgacatccttaggcctgtggctttccaccaaggggccaccctttgaggctcggctgaagagttgctgttttgc
Mutant Sequence: agctcctcccagacattcagggccatcagagtcaccgAGttgcttgaggaataccgaatcatacctgctagtcccatgtgtgggccagcaacatggctcagtgggtaa
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
33848 | AGC TCC TCC CAG ACA TTC AG | Common | A | |||
33849 | TTA CCC ACT GAG CCA TGT TG | Mutant Reverse | A | |||
33850 | GCA AAA CAG CAA CTC TTC AGC | Wild type Reverse | A | |||
33851 | Fluorophore-1 | CCT TAG GCC TGT GGC TTT C | Quencher-1 | WT Probe | ||
33852 | Fluorophore-2 | TGC TTG AGG AAT ACC GAA TCA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
33848 | 0.40 uM |
33849 | 0.40 uM |
33850 | 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 |