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.
Mutant= 138 bp
Wild Type = 130 bp
>chr17:23733540+23733669 130bp AAAGCCTGGACACAGCAGAG ACAACGGTGCATCAGAGACC
Wt Sequence: AAAGCCTGGACACAGCAGAGaCACTGTCCGTGGTTTGGGGGACTACCATTTCCTCTTAGCACTGGACCCTGGTCATAGCACATAGGTCTACCCCACCTCCTCTGTTGCTGGGTCTCTGATGCACCGTTGT
Mutant Sequence: AAAGCCTGGACACAGCAGAGacATCTGGGACTACAGCCAGACCCAGGGTTTTGAGACCATACTGGCTACTACTACCCTGCCTCAGTGTATTCTGTTCATCTCTACAGAATGATGATGCCAACCCTAGTAACCATGGAA
1036 bp deletion beginning at Chromosome 17 positive strand position 23,733,561 bp and ending after 23,734,596 bp (GRCm38/mm10)
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
36971 | AAA GCC TGG ACA CAG CAG AG | Common | A | |||
36972 | TTC CAT GGT TAC TAG GGT TGG | Mutant Reverse | A | |||
36973 | ACA ACG GTG CAT CAG AGA CC | Wild type Reverse | A | |||
36974 | Fluorophore-1 | AGA CCC AGG GTT TTG AGA CC | Quencher-1 | MUT Probe | ||
36975 | Fluorophore-2 | CTT AGC ACT GGA CCC TGG TC | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
36971 | 0.40 uM |
36972 | 0.40 uM |
36973 | 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 |