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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr2:25435759+25435858 100bp AGAGATAAGCTTGACTTCACGAG ACAGAAAAGCTGCCAAGTGC
Mut= 95 bp
Wt= 100 bp
Fam=Mut
Hex=Wt
Mut Sequence:
taaccagttggcctgagaggtcagaggtcactgctaaggggggtgcctcctgtgctttagggttagaggtgcagtggcacttggcagcttttctgt
This mutation is a 2972 bp deletion beginning at Chromosome 2 position 25,432,845 bp and ending after 25,435,816 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44878 | AGA GAT AAG CTT GAC TTC ACG AG | Wild type Forward | A | |||
44879 | ACA GAA AAG CTG CCA AGT GC | Common | A | |||
44880 | TAA CCA GTT GGC CTG AGA GG | Mutant Forward | A | |||
44881 | Fluorophore-1 | TCT AGG TAC TGG TGT GCC TGC | Quencher-1 | WT Probe | ||
44882 | Fluorophore-2 | AGA GGT CAC TGC TAA GGG GG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
44878 | 0.40 uM |
44879 | 0.40 uM |
44880 | 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 |