Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr17:29674341+29674441 101bp GAAGAGGGCCAGGGTGTACT AAATAAACAGCCAGAAAGAGAA
Mut= 102 bp
Wt= 101 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TATCATCTCTGTTTGATAGTAGATAGAACTGAAAGTGAGGTAAGGAAATGGTTATGTGTGCGTTCCTGTTCTGTGGCTTGTTTCATGTCATCAGGAACGTTGATGACATATACGTACGTATACAGCTGGCAAGAGTAGAGACGTCACTGTGACCTCCATTGAGTGCAGGACCCAcactagacataggtgggacatgtggactgtgggtgcatgaggcagtcctgtcatccggacccacctaacgcttctcttcttcttccccagcatctgctacaagcctcagtggatctgacagtgagaccgaggggaagcagccctgctctgatgatttcaaagatgccttcaaagcagattcccttgtggagggaacatcgtcccgatattccatgtataacagtgtttcccagaggcttatggtatgtcttggcttagaatggacttctaaagttgcccaaaagagggagaggaagagggccagggtgtactggtgttggggagtggggtggggcacaagttagCACAGGATATAGGTTCTGAGTATTTTTTTCTCTTTCTGGCTGTTTATTTATTTATTTTGGTTTTTCTTTTTTCTTTTCCTTTTTCTTTCTTTCTTTCTTTTTTTTTTTTTTTCTTTTTGTTTTTTTGAGACAGGGTTTCTCTGTGTAGCCCTAGCTCTCC
This mutation is a 344 bp deletion beginning at Chromosome 17 position 29,674,049 bp and ending after 29,674,392 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44121 | GAA GAG GGC CAG GGT GTA CT | Wild type Forward | A | |||
44122 | AAA TAA ACA GCC AGA AAG AGA A | Common | A | |||
44123 | ACA GCT GGC AAG AGT AGA GA | Mutant Forward | A | |||
44124 | Fluorophore-1 | TGG GGT GGG GCA CAA | Quencher-1 | WT Probe | ||
44125 | Fluorophore-2 | TGC AGG ACC CAC ACA GG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
44121 | 0.40 uM |
44122 | 0.40 uM |
44123 | 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 |