Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr16:10568508+10568623 116bp CTGTCCATTTCTTTTACAATGAGG ATGCCAAAGTCTGTGCTTAGG
Mut = 115 bp
Wt = 116 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
GAGGCCACAGGCTTCTCCCTGGAAGGGCTGGTGGGCCTGCGTAGTGTGTGGTTTCTTAGACAGGTAAAGGTTAAGCCGCTCTTTGTTGCTCACTGGTTGAGTTGTGCTTGttactcggcggcctgtgctggcatttcctccagcccgagctcaccggttgcactttgtgttctctctcctagattatttgctgtctaataactatgtaaactcgatcattgtccataaatttgacttttccgacgaggagatcatggcgtattacatatcgttcctgaaaacgctttcattaaagctcaacaaccacactgtccatttcttttacaatgaggtgagtggcgcccagcaagtgtatgtgtcaagctagtccaagcgtggaggcctTTGCTCTACAAAGCACTTTCCTAAGCACAGACTTTGGCATACTTAAATAAAATTTTTTGTTTTAAAATATGTGGTGTGTGTGTGTGTGTGTGTGTGTGTGTGCGTGT
This mutation is a 274 bp deletion beginning at Chromosome 16 position 10,568,310 bp and ending after 10,568,583 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
50159 | ATG CCA AAG TCT GTG CTT AGG | Common | A | |||
50160 | CTG TCC ATT TCT TTT ACA ATG AGG | Wild type Forward | A | |||
50161 | CCT GCG TAG TGT GTG GTT TC | Mutant Forward | A | |||
50162 | Fluorophore-1 | CCC AGC AAG TGT ATG TGT CAA GCT A | Quencher-1 | WT Probe | ||
50163 | Fluorophore-2 | TGG TTG AGT TGT GCT TGT TGC TC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
50159 | 0.40 uM |
50160 | 0.40 uM |
50161 | 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 |