Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr18:43966735+43966814 80bp AGATTCTTTGAGGTCACTGGTAGC ATCTGATAGAAAAGGTATGGAGGGAGA
Mutant= 125 bp
Wild Type = 80 bp
GTTGTTGTTGTTATATCTGCTGTCACCATAACAAGAAAATTAAATGAAACTATTTTACTTATTGAGTTTTTATAACATGTCAAAGATTCTTTGAGGTCACTGGTAGCATCCATGAaagatcatcaaggatcctttgtctccctccataccttttctatcagattaatgctttttaggtgattaattaattaattaaattaattaattaatttagattaatgcttcctgtttctaacagacaagccagagttattattttttgtgttattgaactgaccaaatagcttctttttctattaaacaaagttgtttgcagtgtttcgatgatataactatatttctttcaggacccatgcatgaaatttcaagcacagatgaaaaatggtacactgacgtgtcccaaaggcaacaattcttctcaaagtctcaatgacatcattttccaaagtgaatgcatcttgtgcaaaagagcactgtgagtaaattccatttccttccacatgttcaaatcaccaactgcctcgtctCTGGAGCATTGGGAACATGGGGGTTTGGGTTTTTTTTTAAATTTTTTATTAGATATTTTCTTTATTTACATTTCAAATGCTATCCCTGTCCTGATTTCCCCTATGAAAACCCCCTCT
A 412 bp deletion beginning at Chromosome 18 position 43,966,767 bp and ending after 43,967,178 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
45132 | AGA TTC TTT GAG GTC ACT GGT AGC | Common | A | |||
45133 | ATC TGA TAG AAA AGG TAT GGA GGG AGA | Wild type Reverse | A | |||
45134 | CAG GAC AGG GAT AGC ATT TGA | Mutant Reverse | A | |||
45136 | Fluorophore-1 | TGG AGC ATT GGG AAC ATG G | Quencher-1 | MUT Probe | ||
45138 | Fluorophore-2 | CAT GAA AGA TCA TCA AGG ATC CTT | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
45132 | 0.40 uM |
45133 | 0.40 uM |
45134 | 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 |