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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr4:133840170+133840285 116bp CAGGCTCACACTCACACCTG CTTACTCTGGTGCTGCTGGA
Mutant= 75 bp
Wild Type = 116 bp
Wt Sequence: caggctcacactcacacctggtctccaccttctcaccctgagtctcactgcttcagGAGCGGAAGATCCTGGACCTGGAGGATTTGGTACAGACCCTCCAGCAGCACCAGAgtaag
Mut Sequence: gcatggcccgCATctatcccaagtcctcgctgtgggtttgtggctgggccctgctctcctatctggaacctctct
A 1340 bp deletion beginning at Chromosome 4 position 134,112,348 bp and ending after 134,113,687 bp (GRCm38/mm10). There is a single bp (A) insertion at the deletion site.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
65934 | CAG GCT CAC ACT CAC ACC TG | Wild type Forward | A | |||
65935 | CTT ACT CTG GTG CTG CTG GA | Wild type Reverse | A | |||
65936 | GCA TGG CCC GCA TCT ATC | Mutant Forward | A | |||
65937 | AGA GAG GTT CCA GAT AGG AGA GC | Mutant Reverse | A | |||
65938 | Fluorophore-1 | CCA CCT TCT CAC CCT GAG TC | Quencher-1 | WT Probe | ||
65939 | Fluorophore-2 | CAA GTC CTC GCT GTG GGT T | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
65934 | 0.40 uM |
65935 | 0.40 uM |
65936 | 0.40 uM |
65937 | 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 |