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.
>chr2:5852041-5852159 119bp CCAGTGATGATTCTGTTTGTAATGG GACAACCAAAGGGACCATGT
Mutant= 105 bp
Wild Type = 119 bp
Wt Sequence: ccagtgatgattctgtttgtaatggagctCAcggtggaagcagcagaggcactctgttccgtccctcgacttcccttctgggcggctgctgcttctgtgacatggtccctttggttgtc
Mutant Sequence: ccagtgatgattctgtttgtaatggagctCCactaaagagaggaaagaaggcagtagaccgtgtccctgccgctccccattgcttcagcacagcagacttagcaa
597 bp deletion beginning at Chromosome 2 negative strand position 5,931,083 bp ACGGTGGAAGCAGCAGAGGC, and ending after TTCCTGCAAAGATCGCTCGC at 5,930,487 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
35904 | CCA GTG ATG ATT CTG TTT GTA ATG G | Common | A | |||
35905 | GAC AAC CAA AGG GAC CAT GT | Wild type Reverse | A | |||
35906 | TTG CTA AGT CTG CTG TGC TGA | Mutant Reverse | A | |||
35907 | Fluorophore-1 | CAG AGG CAC TCT GTT CCG TC | Quencher-1 | WT Probe | ||
35908 | Fluorophore-2 | AGT AGA CCG TGT CCC TGC C | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
35904 | 0.40 uM |
35905 | 0.40 uM |
35906 | 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 |