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= 127 bp
Wild Type = 142 bp
>chr11:96892447+96892588 142bp CTTGCCCTTGAGGCCTTTCT CAGCCAAGGCTACACAGAGA
Wt Sequence: ggagcagatggagtggaacgagtacggagttccagtccaccaacctggacccttgagtggagttagcttctgctaactcggtttacttatgtgtgacctgagaataagacctttctcagggccatttaaagtgGCTGACAAAGGGTCCGGGCAGTGTAAGTTCTGGGTCTTTTTTTTTTTTTCACTGAATTCTTCCTCTCTCTTCTTTACCTCCAGTGTACCTATATCGAGGTGGAACAGGTGGGGAAGACACACGCTGTGATTCTGAGCCGCCCCTCTTGGCTGTGGGGAGCTGAGATGGGGGCCAACGAGCTTGGTGTCTGCATTGGAAACGAAGCAGTGTGGACCAAAGAGCCTGTGGGGCAGGGGGAAGCCCTGCTGGGTATGGACCTGCTCAGGTAGAGAGCTGCTCTTCAGTGCCCCACTTCCTCCCACCTAGCCTGAGTCTATTGGGCTTGTCTCTGTTGGCCTTGCCCTTGAGGCCTTTCTAAGGCGGCTGCAGTCTTATATCTTCCTAGTCTGATAAAGGACCTCCCTTGgcctagagtgctttaatttttttttcagtactttttgttttgagacagggtttctctgtgtagccttggctgtccaggaatttgctctgtagaccaggatggccttgaattcacagagatcctcctgcctgtgtgccaccacacatagttcagtacaacttttgtttgtttgttttttggt
Mutant Sequence: GCTGACAAAGGGTCCGGGCAGTGTAAGTTCTGGGTCTTTTTTTTTTTTTCACTGAATTCTTCCTCTCTCTTCTTTACCTCCAGTGTACCTATATCGAGGTGGAACAGGTGGGGAAGACACACGCTGTGATTCTGAGCCGCCCCTCTTGGCTGTGGGGAGCTGAGATGGGGGCCAACGAGCTTGGTGTCTGCATTGGAAACGAAGCAGTGTGGACCAAAGAGCCTGTGGGGCAGGGGGAAGCCCTGCTGGGTATGGACCTGCTCAGGTAGAGAGCTGCTCTTCAGTGCCCCACTTCCTCCCACCTAGCCTGAGTCTATTGGGCTTGTCTCTGTTGGCCTTGCCCTTGAGGCCTTTCTAAGGCGGCTGCAGTCTTATATCTTCCTAGTCTGATAAAGGACCTCCCTTG
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
37064 | CTT GCC CTT GAG GCC TTT CT | Wild type Forward | A | |||
37065 | CAG CCA AGG CTA CAC AGA GA | Common | A | |||
37066 | CGG TTT ACT TAT GTG TGA CCT GA | Mutant Forward | A | |||
37067 | Fluorophore-1 | CTT CCT AGT CTG ATA AAG GAC CTC CC | Quencher-1 | WT Probe | ||
37069 | Fluorophore-2 | ACC TTT CTC AGG GCC ATT TAA AGT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
37064 | 0.40 uM |
37065 | 0.40 uM |
37066 | 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 |