Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr1:71353716-71353874 159bp AGGTGAGAAATTTGCAAGTCC TAAATTGCTGTCCCTCTTCG
Mut= 131 bp
Wt= 159 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GACTAATTTAAAGTCTCATCTAGTCCATTATCAAAGAATAAAAGTTTACCAGGCAAGAAACCTTATATGCTCATTGGTACATTGGTTAAGTGACCTCATCCTTTAATATTCTAGAAAATAGGTGAGAAATTTGCAAGTCCTTGGAGCCCTGAACCTTCTGCATAATACATTGCTTTAGTCAACTGAAATTAatgtggacaaagtcagagagttcttgctttttcttctctagcgaaattctgaaaaagccgtccaacccgaagagggacagcaatttatcactccggagcacccaagttccagaaagaagtcacacatcattgggtaagccagctctctcttgtaaggcttcgaatagatttttggaacagtcagcaaaagggatactctttaaaaggtttcaatttcacattttcaaataaaaatggagtgcttagattgccccCAACGGACCCCAGGATTGCCCATGGCTGCCTGAATGTGGCAGTATGGTTGTGCAGAATTCTTTCTTCAGAACAAGTAGAAATTAAGTTCTTACTGAAACGCTCTATAGGAAGGAACACCTAGATATTGAGAATTGCATCAAGTCCCAGAGTTGAGTCAGGACTTCACCTGCAAAATAAATAGAACTGTGGTGTATGGAATTTCCATCA
This mutation is a 254 bp deletion beginning at Chromosome 1 position 71,353,549 bp and ending after 71,353,802 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
46911 | AGG TGA GAA ATT TGC AAG TCC | Common | A | |||
46912 | TAA ATT GCT GTC CCT CTT CG | Wild type Reverse | A | |||
46913 | AAT TCT GCA CAA CCA TAC TGC | Mutant Reverse | A | |||
46914 | Fluorophore-1 | AGC GAA ATT CTG AAA AAG CCG | Quencher-1 | WT Probe | ||
46915 | Fluorophore-2 | CTG AAA TTA CAA CGG ACC CCA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
46911 | 0.40 uM |
46912 | 0.40 uM |
46913 | 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 |