Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr3:69094565-69094671 107bp CAAGCAGTGGTTCAGTCCAG CACTATGCCTGGCCAACAC
Mut= 92 bp
Wt= 107 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
AAGGAAGGAAAAGGTTATGGTATAGAATTGAAAGTTAAAATCAAAGTAAGAACCAAAAACAAACTTGAGACTGATTTTTAACTTGTGAGCTTTGAAAACTGGCACAATGAGTAATTGAAAACTGAGGAGTAATATGAAAGTACATTTCTAAGTCAGgatgtcggactttcacactgaaatggttgggctatgtttttaaggctagaaaacatttcacatactagaggggagaaattgttggtagacgtttagatgatatgagacgtatggttttaaaaaaatcacttcttctttggtagtccttctttacagtttgaagctgcgtgggctttgacaaacattgcatctggaacgtctgaacaaacgcaagcagtggttcagtccagtaagttgttaattaacaaagttgtaattgttttccttcccattggtagaaaataaactaccagGCGTAGTGTTGGCCAGGCATAGTGGCTCATGCTTTTAATTCCAGTACTTGAGAGGCTTACATAGGAGGATCATGAGTTTGAGGCCTGCATGGGCTACGATACTGAGACCTCA
This mutation is a 293 bp deletion beginning at Chromosome 3 position 69,094,589 bp and ending after 69,094,881 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
41184 | CAA GCA GTG GTT CAG TCC AG | Wild type Forward | A | |||
41185 | CAC TAT GCC TGG CCA ACA C | Common | A | |||
41190 | GCT TTG AAA ACT GGC ACA AT | Mutant Forward | A | |||
41191 | Fluorophore-1 | AAG TTG TAA TTG TTT TCC TTC CCA | Quencher-1 | WT Probe | ||
41192 | Fluorophore-2 | TGA AAG TAC ATT TCT AAG TCA GGC G | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
41184 | 0.40 uM |
41185 | 0.40 uM |
41190 | 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 |