Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr3:133320859+133320973 115bp TCAAAAAGCAGCCCAGGAC GTCGTGGAAAGGGGAGATGT
Mut = 116 bp
Wt = 115 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
AACTTTTAAGGTGTGAAGTTAGCTTTGCAAAAAGCACACAAGTTCAAAAAGCAGCCCAGGACCTCCTTTCGATAAAGAGCAGCGCGCgcggcggtaactctgtgcctattttctttacagagcatgtagctggtcattacatctcccctttccacgacattcctctgaaggcggactgtaaagaggtattgtttttacttgtttgggggatgaagaacttcctgtacacgatatatcaaaatgcttcactattgcccaaaagatgcctttaaagtgaagatgtggttggcaggcaggggaagaaaccaagGAGCTAGAATGGGGCAGTAAGCCTCTGTTCTAAATTAAAATTTGTATGAGGCCAGAAGAAAGGGAGGCATTTACAGACTATGAAGTTTAGACTGCAGTGAGAATTGAAGTAATATGGGA
This mutation is a 223 bp deletion beginning at Chromosome 3 position 133,320,903 bp and ending after 133,321,125 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
51030 | TCA AAA AGC AGC CCA GGA C | Common | A | |||
51031 | GTC GTG GAA AGG GGA GAT GT | Wild type Reverse | A | |||
51032 | AAA TGC CTC CCT TTC TTC TG | Mutant Reverse | A | |||
51033 | Fluorophore-1 | CGG TAA CTC TGT GCC TAT TTT CTT T | Quencher-1 | WT Probe | ||
51034 | Fluorophore-2 | ATG GGG CAG TAA GCC TCT GT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
51030 | 0.40 uM |
51031 | 0.40 uM |
51032 | 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 |