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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 114 bp
Wild Type = 97 bp
>chr5:3622623+3622719 97bp AAGATGAAATGAAAATTAGACATCTTT GCTCCTGGTCACTAAGTGCT
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
48844 | AAG ATG AAA TGA AAA TTA GAC ATC TTT | Wild type Forward | A | Wt F | ||
48845 | GCT CCT GGT CAC TAA GTG CT | Common | A | Common | ||
48847 | Fluorophore-1 | TTG GTG AAG CAT CCC TGC | Quencher-1 | WT Probe | ||
48849 | TTC ATT TAC TTT ATA AGA TCC AGG AGT | Mutant Forward | A | Mut F | ||
48850 | Fluorophore-2 | ACC TCC TAC ACT TCA GGA CCT TAT G | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
48844 | 0.40 uM |
48845 | 0.40 uM |
48849 | 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 |