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= 100 bp
Wild Type = 90 bp
>chr11:108784561+108784650 90bp GCAAAACAAAATCCAAACTCAG AGCTGCTGCTGGGATCTG
Wt Sequence: cttttctctctcccccctcccccctccttttctttccagAGAGGAGGCTCACATGAGCCCCTGCTGACTTAAGAGAGACCAAGCCGATTGCTGAGAGGAACTGGAAGAAGAAAAAGGAGGAGGAGGGAAAAAAAGCAAAACAAAATCCAAACTCAGTGAGACGCTCTCCCTCACCATGAGtaGCGCCGTGTTAGTGACTCTCCTTCCAGATCCCAGCAGCAGCTTCCGCGAGGATGCTCCGCGGCCCCCGGTTCCGGGAGAAGAAGGGGAGACCCCACCGTGTCAGCCTAGTGTGGGCAAGGTCCAGTCCACCAAACCTATGCCCGTTTCCTCT
Mutant Sequence: TTCCAGAGAGGAGGCTCACATGAGCCCCTGCTGACTTAAGAGAGACCAAGCCGATTGCTGAGAGGAACTGGAAGAAGAAAAAGGAGGAGGAGGGAAAAAAAGCAAAACAAAATCCAAACTCAGTGAGACGCTCTCCCTCACCATGAGTgtcgagtatcggaaatgtcttcaggctggaatgaaccTTGAAGCTCGAAAAACAAAGAAAAAAATCAAAGGGATTCAGCA
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
34714 | GCA AAA CAA AAT CCA AAC TCA G | Common | A | |||
34715 | AGC TGC TGC TGG GAT CTG | Wild type Reverse | A | |||
34716 | GTT TTT CGA GCT TCA AGG TTC | Mutant Reverse | A | |||
34717 | Fluorophore-1 | AGT ATC GGA AAT GTC TTC AGG C | Quencher-1 | MUT Probe | ||
34718 | Fluorophore-2 | CAT GAG TAG CGC CGT GTT AG | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
34714 | 0.40 uM |
34715 | 0.40 uM |
34716 | 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 |