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.
>chr19:5457322-5457479 158bp GGAGCCTGAGAGGCAACC GCACCAGCTCCTCCTTACC
Mutant= 136 bp
Wild Type = 158 bp
Wt Sequence: GGAGCCTGAGAGGCAACCGGCCCGAGGAGGGGCTCCAGAGCCTCGGCCGTGGGCGGGCGATGCTGCAGCTGCCTGATCAACTATGGAggCCGAAGCAGGCGGCCTGGAGGAGCTGACGGACGAGGAGATGGCGGCGCTGGGTAAGGAGGAGCTGGTGC
Mutant Sequence: GGAGCCTGAGAGGCAACCGGCCCGAGGAGGGGCTCCAGAGCCTCGGCCGTGGGCGGGCGATGCTGCAGCTGCCTGATCAACTATGGAggTCGTAGCTTCGGCTGAGCACCAGGGCGCTGGAATGGACTGGAAAAGC
680 bp deletion beginning at Chromosome 19 negative strand position 5,457,391 bp, GCCGAAGCAGGCGGCCTGGA, and ending after GCTGTGGACCTCCAGACCTG at 5,456,712 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
35171 | GGA GCC TGA GAG GCA ACC | Common | A | |||
35172 | GCA CCA GCT CCT CCT TAC C | Wild type Reverse | A | |||
35173 | GCT TTT CCA GTC CAT TCC AG | Mutant Reverse | A | |||
35174 | Fluorophore-1 | TAG CTT CGG CTG AGC ACC | Quencher-1 | MUT Probe | ||
35175 | Fluorophore-2 | ACG AGG AGA TGG CGG C | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
35171 | 0.40 uM |
35172 | 0.40 uM |
35173 | 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 |