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.
>chr7:66709084+66709193 110bp TGACATGTGGATATTTTCATAGTTG TGCTCTGACAAAACAAAGAAGG
Mutant= 99 bp
Wild Type = 110 bp
Wt Sequence: tgacatgtggatattttcatagttgtttttgaTCcttcacttctatcctgtgttgcctttcctgccttaaagagtattagaatattttccttctttgttttgtcagagca
Mutant Sequence: tgacatgtggatattttcatagttgtttttgaTGtgtggtgtatgtattgggtgtatgaacgtatgcgtgtggatatgtgtgtgcttgtagaggctaga
466 bp deletion beginning at Chromosome 7 positive strand position 66709117 bp for 197 bases where 3 endogenous bp (GGT) are retained, followed by an additional 269 bp deletion ending at 66709585 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
36253 | TCT AGC CTC TAC AAG CAC ACA CA | Mutant Reverse | A | |||
36254 | TGA CAT GTG GAT ATT TTC ATA GTT G | Common | A | |||
36255 | TGC TCT GAC AAA ACA AAG AAG G | Wild type Reverse | A | |||
36256 | Fluorophore-1 | CTA TCC TGT GTT GCC TTT CCT G | Quencher-1 | WT Probe | ||
36257 | Fluorophore-2 | TGG TGT ATG TAT TGG GTG TAT GAA C | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
36253 | 0.40 uM |
36254 | 0.40 uM |
36255 | 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 |