Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr3:90543152+90543251 100bp CTCTTGGCTGCTTCCTTTGT GCAAGTCCCTACAGCCTCAG
Mutant= 97 bp
Wild Type = 100 bp
AGCCCCTGCTTTCCTCTGTCTTGATTCTTCCCAGCTAAGGTTGCCACAGAACAAGGGACCTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTAGGGGGCAACAGTTACCTGCTTCCTCTCCTCACAGGAGCCTCCTGCTCTCAGAGGGCTCTGACAGGCATCAGGCCTGCCTGGGGCAGGGGAAGGGCCTGGAGAACCTGTCTTGgcagttggcagtctggctggggaacacctggattagaggagtcacatagctctccttccaggccacccccacacccactttgctatcctatgtctgctttctcccttctctgaaaggcaagaagtctgaggatgctgtctgtgtgcagggatgtcccttctcatctctcttttctgcttcctaggcccagagaggagctgaggcagtgagatggctgactgctatacagagctggagaaggcagttgttgtcctggtggaaaacttttataaatatgtatccaagcacagcctggtcaaaaacaagatcagcaagtccagcttccgaaagatgctccagagagagttgaaccacatgctgacggtatccccactgctgccctctgtgcctgctcttctcctcacagctctctcagtgacccctccctccctgtgctctgctccgtgttccctcctagcctgctagcctcctctgccctgctagcctggccaagggactctgcctagggtcctgtctgggccatcccaagctttactgattcaagttcttccacaggacacagggaaccgaaaggcagctgacaagctcatccagaacctggatgccaaccacgacgggcgcatctgctttgacgaatactggaccatgattggtggaatcaccagccccatggccaacctcatccgccaacaggagtgccagcaggagagccagcaggagtgccagcaggagagccagcaggagagccagcaggagagccagcagggcagcagctagaggcccctcgtgggccccttttcatgtcctgacccatgtcctgccagtgttctttccatgctttaagttgactctttcttcctcccttctccctccagacctgttcctacccttgctgaacagcacaagatgggtgggtcgggggctatcaaggccactgtggtagtgcttccctgagcattcaatccctggatcctgtaggccctgggagggggtcccctttgtgaaatatcaatgctgtttggggccctccccccaagacttctctcacttgttgagtctgtgtgctctcgcaatgttcctgtctaattcttgactgattctgggtctgtcctgacctctagaggtgagcttgctgcttgaggtctcccagctcctgactgtggcagctgtggtagtggcagtaccagaattagccgcttcttttcctcccaacctcttagccgggtagagaggcattcccaggggctcctgtccagctgtctctgtctgggaatgatctaacctgggttgagcctcctctctgctctgccaccgaaataaacgttgaatttggagtttgtaactcttggctgcttcctttgtctttaattgctccacacttcttgggaatctattctaccttggtgATCTTTGTCCTGGACCCTGAGGCTGTAGGGACTTGCATAAGAAAACATTACATGCAAACAAAATAAGAAGAAGTTAAACTGGGATTGATGGCCCGCATGTATAATTCTTGAATCTAGGCTGATGTATTAGGGGTCTGGAGTTCAAGGCCAGCCAGAGATGCATAGCTCAGCATGGTGGCGTTTGCCTGCCCAAAGGCAAAAAGAATAGGTTATATTCTCAGATTCAAGTTCAACTTTTAGGTGCATAGTAGGTGT
A 1406 bp deletion beginning at Chromosome 3 position 90,541,810 bp and ending after 90,543,215 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
45960 | AGG GCT CTG ACA GGC ATC AG | Mutant Forward | A | |||
45961 | CTC TTG GCT GCT TCC TTT GT | Wild type Forward | A | |||
45962 | GCA AGT CCC TAC AGC CTC AG | Common | A | |||
45963 | Fluorophore-1 | TGC TCC ACA CTT CTT GGG A | Quencher-1 | WT Probe | ||
45964 | Fluorophore-2 | AGA ACC TGT CTT GAT CTT TGT CCT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
45960 | 0.40 uM |
45961 | 0.40 uM |
45962 | 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 |