Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr2:120283677-120283801 125bp TCAGGTCAAGGTAAATGTCAC TGAGTTCTGGGAGTGCTGGA
Mut= 104 bp
Wt= 125 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
CTGACCGCTGAGGGCTTGGGGGTCTCTTTGCAGGAGGAGGCCTCTGTCTTCTGCCGGCTCACTGTGAAAATCCTGGAGGCTCGGAGTCTGCCCAGGGCTGACCTGTGTGAGTGACTGTGGGTGGGGCCTGGATCCCAAGGGAAAGGACCAAATGATCTTTGAGGTGGGGCTTAGCTGCTCTCAGGGGATACTGGGGTTGTCAAAGGAACCTGGACCAAATCCCTGGAaaggatgtatagaatccactctcttctgatgcctggtccccttgactttgtcagtgagtcaggcagatccctatgtgactgtgcagctgccaacagcttctggaatgaaattcaagacccaaacagtcactaactccagtcatcctgtgtggaacgagaccttcagtttcctcatccagagtcaggtcaaggtaaatgtcacgtgggtgtcgcaccagccactcactcgtagaacagtccatcccccttccctccacatgaggaccttgggaacaaGTGGGATTGCTCCAGCACTCCCAGAACTCAGGGGAGGAGGGTGTCTGTGCTCCAGACTCTATGTGCTGTGAGCAAGAATGCGGCCCTGCAACTCCCTGGCTGTGGGACTGACGTTTGGCAAGCTGAAGCTTCTGGGGAGCCCATCTCTGATCTGTGGATAGAAAGCATTCCTTTTTAATGGGGCTGCTGCAGGGCTGAAGGGAAATTACATAGAAAGGGATCGCAAAGTA
This mutation is a 276 bp deletion beginning at Chromosome 2 position 120,283,707 bp and ending after 120,283,982 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
47698 | TCA GGT CAA GGT AAA TGT CAC | Wild type Forward | A | |||
47699 | TGA GTT CTG GGA GTG CTG GA | Common | A | |||
47700 | GAT CTT TGA GGT GGG GCT TAG | Mutant Forward | A | |||
47701 | Fluorophore-1 | ACC AGC CAC TCA CTC GTA GAA C | Quencher-1 | WT Probe | ||
47702 | Fluorophore-2 | TGG ACC AAA TCC CTG GAG T | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
47698 | 0.40 uM |
47699 | 0.40 uM |
47700 | 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 |