Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr2:110021681+110021791 111bp TGCTGAAGTTGAGGCAGATG TCTAACAGTCTCCTTTCTACAAATCC
Mut = 105 bp
Wt = 111 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
ACTGTTAAGGAAGGAAAAAAGGTCTGGGTTTGATTACAAGTAGTAATTAACTGTTCAAGTAGGATTTTTCAGAATAAAAAAGAATCATCAATGCTGAAGTTGAGGCAGATGCATGGGGTTTGGGATGCTCCCACGGATtcttacaaaggcagtttgatgggtgaagtcattcagatggatttgtagaaaggagactgttagaagtattacattgttgctttcattgtacagctgcaggtttcccagtgggaagagttctcggtagtaagtgctcccattctctttctcgtagctgtgaagttgagtcgcaagacaaacaagagaagctggtgctgttacagcacggcagtctgacgccatgggagatgtggtttgttggcaaggagaaagaagagcgaggccggctacaacagaaatttctggaggtaaactaaacagtgtctcttcattgacccctgggcccacagagatccacctgtctctgcttcctcagtgctggaatcaaggttgtgcacagccatccccagccaaccgttctattttagtcgtcatattcaaatgtagccacagaatactaaaGGGGACTCTTGTGAAGGAAATAGAATCCAACTCTTATAACCTGCATTTTCTCCTGTGAGATATTAATATATTTCCATATCATTGAAAACATGTTTTTGTTTGTTTTGTTTG
This mutation is a 441 bp deletion beginning at Chromosome 2 position 110,021,728 bp and ending after 110,022,168 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
50601 | TGC TGA AGT TGA GGC AGA TG | Common | A | |||
50602 | TCT AAC AGT CTC CTT TCT ACA AAT CC | Wild type Reverse | A | |||
50603 | TCA CAG GAG AAA ATG CAG GT | Mutant Reverse | A | |||
50604 | Fluorophore-1 | CAG TTT GAT GGG TGA AGT CAT TCA | Quencher-1 | WT Probe | ||
50605 | Fluorophore-2 | CGG ATG GGG ACT CTT GTG AA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
50601 | 0.40 uM |
50602 | 0.40 uM |
50603 | 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 |