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= 105 bp
Wild Type = 142 bp
>chr1:163081235+163081376 142bp CTTCTGCAGCCCACTCTCA TGCAAGCTGCATACAACAGA
Wt Sequence: TTTGATGCTAGGTATTTTTTTTTTTTTTTTTTGGAGTTAATGGAGCCAGAAGAAACTCTAGAATCGGAGAGTTCAGAAAAGTCACTATTTTCCTCACAGCAAGAGTATGAAGAGTCGCAAGAAGCAGAAGAAACAGGCGCTGAGAACCCCCTTCTGCAGCCCACTCTCACTGGGGACGTGGAAGGTTTGCAGAAGATTTTTGAGGACCCTGAACATCCTCACCATGAGCACGCTGTGCAGCTGCTTTTGGAAGAAGACATTGTTGGGAGAAATCTGTTGTATGCAGCTTGCATGGCTGGCAAAAGTGACGTAATTAAAGCCTTGGCAAAGTATGGGGTGAATCTGAATGAGGCAACTGCCAGAGG
Mutant Sequence:aaaagaaaaaggaaaaaagAAaacaccaccaccaccaaaaaacCCAAGTCCCTGCATCCCACAagactagtttagtgtagttccctctgctgaaagattaagtaagcaggggttgggatgtagctcagttggtacaacttgtac
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
31240 | CTA CAT CCC AAC CCC TGC T | Mutant Reverse | A | |||
31241 | Fluorophore-1 | TGT AGT TCC CTC TGC TGA AAG AT | Quencher-1 | MUT Probe | ||
36385 | GAA AAC ACC ACC ACC ACC A | Mutant Forward | A | |||
36386 | CTT CTG CAG CCC ACT CTC A | Wild type Forward | A | |||
36387 | TGC AAG CTG CAT ACA ACA GA | Wild type Reverse | A | |||
36388 | Fluorophore-2 | CCC TGA ACA TCC TCA CCA TGA G | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
31240 | 0.40 uM |
36385 | 0.40 uM |
36386 | 0.40 uM |
36387 | 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 |