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.
>chr9:40015664+40015782 119bp CCCAGGTGATGAGATTAAAGG GGAAACTCAAGCTGCAAAGG
Mutant= 121 bp
Wild Type = 119 bp
Wt Sequence: cccaggtgatgagattaaaggttgaaaGAagggggaaattagaagagtcctagggtgtcccctgccttgtctttgtaaagataactggttcttcgtgtgcctttgcagcttgagtttcc
Mutant Sequence: cccaggtgatgagattaaaggttgaaaGAtcagcttctcgactctcttagcagagagagtaagtagttaaggacactttgtgttcactctagtagaggaggctcagcctctggcatttcat
652 bp deletion beginning at Chromosome 9 positive strand position 40,208,107 bp, AAGGGGGAAATTAGAAGAGT, and ending after GACAATTCTGCCCACTTTAG at 40,208,758 bp (GRCm38/mm10)
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
35230 | Fluorophore-1 | AGC TTC TCG ACT CTC TTA GCA GAG | Quencher-1 | MUT Probe | ||
35231 | CCC AGG TGA TGA GAT TAA AGG | Common | A | |||
35232 | ATG AAA TGC CAG AGG CTG AG | Mutant Reverse | A | |||
35233 | GGA AAC TCA AGC TGC AAA GG | Wild type Reverse | A | |||
35234 | Fluorophore-2 | AGT CCT AGG GTG TCC CCT G | Quencher-2 | Common |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
35231 | 0.40 uM |
35232 | 0.40 uM |
35233 | 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 |