Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr12:105675042-105675138 97bp GCCTTGCGTTACATTGTCAG GAAGTCAGACAAACAGAAACACAGA
Mutant= 105 bp
Wild Type = 97 bp
CCAGTGCTTTTAACCACTGAGCCTTTTTCTCCAGCCCAGCCTTGCGTTACATTGTCAGTGCTCAGCAcctcggtgggcatcttcattcacatcattgaaaaagttgacattctgtgtttctgtttgtctgacttcaaaatgagtctgtttttattctctctagtgtctcaatgaaatcctggagtcagcagatgcaccattagaagtcactgaaggatttattcagtccatttccctgtccgtcccatggggctccttgctgcaggacaactgtgccctggaagtgagaggactggagatggtcttccggcctcgacctcgagtaggtaggtgtttcctggtgcccgttaacccagctGTAAGCAAGTGAGCGTTGGATTGATGACTTTCAACTGGGTCTGTGAGACTTTTCAGTTGCTAGGATACGAGGATTTAGCACTGTAGGATGTATGTGAGTATGCCTATA
A 291 bp deletion beginning at Chromosome 12 position 105,674,819 bp and ending after 105,675,109 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
45122 | GCC TTG CGT TAC ATT GTC AG | Common | A | |||
45123 | GAA GTC AGA CAA ACA GAA ACA CAG A | Wild type Reverse | A | |||
45124 | AAA TCC TCG TAT CCT AGC AAC TG | Mutant Reverse | A | |||
45125 | Fluorophore-1 | TCG GTG GGC ATC TTC ATT | Quencher-1 | WT Probe | ||
45126 | Fluorophore-2 | AGC AAG TGA GCG TTG GAT TG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
45122 | 0.40 uM |
45123 | 0.40 uM |
45124 | 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 |