Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mut = 124 bp
Wt = 118 bp
Fam = Mut
Hex = Wt
>chr12:33342244+33342361 118bp AGTCCCATCTGTAACCAGTGA GCACCATCTCCCAGGTACTC
Wt Sequence (deletions in lower case):
CACCGTGCCAGCCTCTCACCCCCTGTTGGTCCTTTACCCGGCACAGATCTCCCTCCCATCAAAACGGGGAATCACGCTGCTCGCTTACAGCAGGAGCATAAATAAGTGATAAGGAAGTGTTGTGATAACGATAAgcgggggtgtagctcctcggtggaccagatctatcaaagaaggcggtggtgcatgtgtgttcctttatttttcagcgttccagttcccgtagtcagtttagagaaaattcctaacctagtgaaggcagatggtgccaatgtcaaaatgaactctacaaccactactgcagccacttcctcctcctctgccgtctccactccgcctttaattaagcccaccttgatgtccaagtcagtgccaccttcaccagagaagatcttaaatggcaaggggattctgtcagccaccatagacaagaagcacccaaacggcaccaaaaacagcaacaagccttaccggagactttcaggtatgtgttcctgtggttagagtctggctcacggtaagatgacaactgagtcccatctgtaaccagtgatgtgttcgtagaggcaaacagctgtttggtttgaacagcgatctggagtgtagctgcaaGTAGGACCGGCACCCCGAGAGTACCTGGGAGATGGTGCTCTCATCCCCCAGTGCAGATAAGGTCCCCAGCCAAGTTTTGA
This mutation is a 478 bp deletion beginning at Chromosome 12 position 33,391,845 bp and ending after 33,392,322 bp (GRCm39/mm39).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
57967 | GCA CCA TCT CCC AGG TAC TC | Common | A | |||
57968 | AGT CCC ATC TGT AAC CAG TGA | Wild type Forward | A | |||
57970 | Fluorophore-1 | ACA GCG ATC TGG AGT GTA GCT G | Quencher-1 | WT Probe | ||
58737 | CTC CCT CCC ATC AAA ACG | Mutant Forward | A | |||
58738 | Fluorophore-2 | CAC GCT GCT CGC TTA CAG C | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
57967 | 0.40 uM |
57968 | 0.40 uM |
58737 | 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 |