Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr8:105338657-105338765 109bp CCCAGCTCAGGTATCAGCA ACCCCCATTTCCACATAATTG
Mut = 98 bp
Wt = 109 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
CTTCCTTGAAGGCAGCCACTTCCCTCTCTCCTCCTCCATGGCCCTTCCTTATTTGTGGTTTGTTAATGCAAAAATAAAAAGCAAAAACCAACCAACCCCAACCCCCAAAataccctccagggctgtagcttgatggtttaacatgtgggcttgattcctagtagcacagaaacaaaacagacggcaaaaatctgagctgcagcaccagccctgatagttctgtccctttgtttccagctggaggactgtgcattgcaagtgtctccctccggatactacctggacccggagctgtccctagaagaacagcgggagatgctggagggtttctatgaagagatcaggtcagagttgaaagggtgagggaggccagggaacccttcccatttgaggaggtctggccagagccttcaaccccgggccccactggggcccagctcaggtatcagcagctgagatactctgtgtacagcctgggctgtaggccagtgcagagaacaatgccgggggctgGTACCCACAATTATGTGGAAATGGGGGTACTCTCATACCTCAGTACCTGTGTTTATAAAAACTCTCCTGTTCGGCTA
This mutation is a 404 bp deletion beginning at Chromosome 8 position 105,338,685 bp and ending after 105,339,088 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
51020 | ACC CCC ATT TCC ACA TAA TTG | Common | A | |||
51021 | CCC AGC TCA GGT ATC AGC A | Wild type Forward | A | |||
51022 | GGC CCT TCC TTA TTT GTG GT | Mutant Forward | A | |||
51023 | Fluorophore-1 | CTG TGT ACA GCC TGG GCT GTA | Quencher-1 | WT Probe | ||
51024 | Fluorophore-2 | AAC CCC AAC CCC CAA AGT A | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
51020 | 0.40 uM |
51021 | 0.40 uM |
51022 | 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 |