Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 100 bp
Wild Type = 87 bp
>chr17:36872134+36872220 87bp CTGAGGAGCAGGCAGGAGTA AAATTCCCACTTCCCTCTTGA
Wt Sequence (deletions in lower case):
CTGAGGAGCAGGCAGGAGTAGGGGGCTCTGATTTTACTgcctaaccttgggaggtgtgggcatagatcaagagggaagtgggaatttataaggaaagggaagaaaggacagatgagatgccacaggaagagggggaaaagatgggagaagatctcgctgtgaggagagtaaaatgttctccctggctctcatcttcccttctcagactcaggtggccaccaagaggcaacaggtgatctcacagtttgcgcatctgagccagttcctgcagcaacagcagactgccctcttagcacaactggaggggttagacggggacatcttgaagcagcaggaagagtttgattccctggccactggggagatctgccggttcagcaccctgattgaggagctggaggagaagaacaagaggacagccagggggctcctgacggtgaggcctggcctccagggtctctggaattactgcctaattggtacctccttgacatacatgtttaggcactgaagatggaggaggtacatggggaaagggggttggattgtaatctgtgcttccccaagagctgcctggtcactgtgctctctgttgttggtctctccttcctgccctccgtagaatggagaggaaatgaggatatgacagtaaaaggaggtggaagttgctcctgagacgtatcacacactgtgcaaacaagtgccaggtgttgcctccaaagcaaaactttcccctccatggccaggaagttcttcctagactgtaccacttccttgaactggaggagagttattggggttccaggagcatgctttagggagatgcactgggtactgggaaataagagactttggtgtagaacttgctcacttttgcctatagctggggagaagtaggtcgtatcatcaactctgtccatgaaacaaaggtctgggttctggtcttaatgtccaaggctgggaatacggcagtattttgggttttatggtcataacaaaagtgggagagaaaaactagaacctaattctctctctctctctctctctctctctctctctctctctctctctctctctctctgtccatctgttcctccctctctccctccctcctttcctttcttcctttcccctctcaggatatcagaagcactctaataaggttagtgataaagtatctacttttttcttccccatagacattcatccatacaccacgtacctagttattgacgtgaaaataaaccactttctcagtttagggtacagtcatatataaatgtacaaatgctcaaatgtgttctcagatagtgctgtagatctacccacttgtgcagcagtggggtccatctgagcggatccaagtctctatgtgggaagttgctcctcaagGCCTTGTTAGAAAAAGATGGCTGTCTGGGGACATCATTAGAAGACAATAATTCTGGGGCTGG
This mutation is a 1354 bp deletion beginning at Chromosome 17 position 36,872,172 bp and ending after 36,873,525 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
65358 | CTG AGG AGC AGG CAG GAG TA | Common | A | |||
65359 | AAA TTC CCA CTT CCC TCT TGA | Wild type Reverse | A | |||
65360 | CCA GCC CCA GAA TTA TTG TC | Mutant Reverse | A | |||
65361 | Fluorophore-1 | CTA ACC TTG GGA GGT GTG G | Quencher-1 | WT Probe | ||
65362 | Fluorophore-2 | CTC TGA TTT TAC TGC CTT GTT AGA AA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
65358 | 0.40 uM |
65359 | 0.40 uM |
65360 | 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 |