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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr19:41924549-41924709 161bp TAAGTTCTTGCTCCCAAGTCC ACATCTTTAGCACTGGGTGTTAC
Mut= 160 bp
Wt= 161 bp
Fam=Mut
Hex=Wt
MUT Sequence:
aaccccctcctctgacctctgtagacagcagacacacatgtggtacacacacatacatgtaggcaaaacactcatcgcatcagataactctaaaaataaaagcaaaaccaagcaaagacccatggtcccctacaattgtaacacccagtgctaaagatgt
This mutation is a 3625 bp deletion beginning at Chromosome 19 position 41,924,589 bp and ending after 41,928,213 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
48293 | TAA GTT CTT GCT CCC AAG TCC | Wild type Forward | A | |||
48294 | ACA TCT TTA GCA CTG GGT GTT AC | Common | A | |||
48295 | AAC CCC CTC CTC TGA CCT CT | Mutant Forward | A | |||
48296 | Fluorophore-1 | ACC ATG GCT ATC TGG GAA GC | Quencher-1 | WT Probe | ||
48297 | Fluorophore-2 | ACC AAG CAA AGA CCC ATG GT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
48293 | 0.40 uM |
48294 | 0.40 uM |
48295 | 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 |