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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr5:31201564-31201674 111bp AGGCCAAGATCTGGAGTTTGA CCACGACCCTTAAACACACC
Mut = 110 bp
Wt = 111 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
GAGATTCCACAAGCACCCTCCCCTCTTTAGTGGATACTGAAGATTCCTTCGACgaaggtcctggggccctggtgttggagagcgatttgctactaggccaagatctggagtttgaagaggaagaggaagaggatgaaggtgacggccacaacgaccagctcATGGGCTTTGAGAGAGACTCTGAAGGTGTGTTTAAGGGTCGTGGTTTAGGATTAGTGGCAATTGGT
This mutation is a 108 bp deletion beginning at Chromosome 5 position 31,201,608 bp and ending after 31,201,715 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
53570 | CCA CGA CCC TTA AAC ACA CC | Common | A | |||
53571 | AGG CCA AGA TCT GGA GTT TGA | Wild type Forward | A | |||
53572 | ACT GCT TGG GAG GGA GAT TC | Mutant Forward | A | |||
53573 | Fluorophore-1 | AGG AAG AGG ATG AAG GTG ACG | Quencher-1 | WT Probe | ||
53574 | Fluorophore-2 | CAC CCT CCC CTC TTT AGT GG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
53570 | 0.40 uM |
53571 | 0.40 uM |
53572 | 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 |