Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr6:41355159+41355287 129bp GGTAACCCATCAAAATGTAGTCAC CCCAACATAGAGAGACTGTAGAGGA
Mutant= 106 bp
Wild Type = 129 bp
Wt Sequence (deletions in lower case):
TGGTAACCCATCAAAATGTAGTCACATACAGAGTACTTTGCTACTGTAAATTTAAgataataatgtccagcataatgagaattttaactttcaatttaggagtattcctctacagtctctctatgttgggcaaacatttattttttcctacagggatacagtgttaccttcttgccttctttcttgccttctattattgcactctttaaggatgcctagtcagaggcctgaaggagtcaccttccagcaccttcatgaattttctcctcaatgatcttttttttttaattgaagttgctttccctgtggatgatgatgacaagatagttggaggatacacctgccgagagaattctgttccctaccaggtgtccctgaactctggctaccacttctgtggaggttccctcatcaatgaccagtgggtggtgtctgcagctcactgctacaagtcgtaagtggtggtccctaatGGTACAGAATTCAAATTTGGCCTTTCTGCAGACACAGTATAAGATCAGGCAAGA
A 418 bp deletion beginning at Chromosome 6 position 41,355,213 bp and ending after 41,355,630 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
49687 | GGT AAC CCA TCA AAA TGT AGT CAC | Common | A | |||
49688 | CCC AAC ATA GAG AGA CTG TAG AGG A | Wild type Reverse | A | |||
49689 | TTG CCT GAT CTT ATA CTG TGT CTG | Mutant Reverse | A | |||
49690 | Fluorophore-1 | TGT CCA GCA TAA TGA GAA TTT TAA CTT | Quencher-1 | WT Probe | ||
49691 | Fluorophore-2 | TGC TAC TGT AAA TTT AAG GTA CAG AAT TCA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
49687 | 0.40 uM |
49688 | 0.40 uM |
49689 | 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 |