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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mut= 137 bp
Wt= 132 bp
Wt Sequence (insertions with carrots ^g^ (g insertion)):
GACCTTGGCTTCATTCTCCAGTACTATGTGGGCGCTGTTACTAACAGGTATATGCTATCATTGGTTTCCATAG^ATAACTTCGTATAATGTATGCTATACGAAGTTATACTAGT^CTAAGCATCAGGTACATTGTAGATGAATGTCCTTACACCAAAATTCAAAAACATCTTTT
Mutation details
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
39617 | Fluorophore-1 | TGG TTT CCA TAG CTA AGC ATC AG | Quencher-1 | WT Probe | ||
39624 | ACT ATG TGG GCG CTG TTA CT | Common | A | |||
39625 | ATC TAC AAT GTA CCT GAT GCT TAG ACT | Mutant Reverse | A | |||
39626 | Fluorophore-2 | TCA TTG GTT TCC ATA GAT AAC TTC G | Quencher-2 | MUT Probe | ||
39690 | AAA AGA TGT TTT TGA ATT TTG GTG | Wild type Reverse | A |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
39624 | 0.40 uM |
39625 | 0.40 uM |
39690 | 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 |