Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr3:152128442-152128529 88bp CAGGGGTTTTAGAAGGATGTGA CAACCCAGTCACCTAAGAAGG
Mut= 82 bp
Wt= 88 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TGCTGCCCCTCCGGGGGACATGTTTGGTTTCCAGCACTGCAGCCCTCTCTAACTTCATCTGCAGGGGATCTGATGCCCTCTGTAGATTCTCACAGGGGTTTTAGAAGGATGTGATGTGGCAGGTGTGACAGGTACgcagagggaagaacagtgtctcacccttcttaggtgactgggttggaggatgctggggaaagatgtctagaggaaatggtagctgtgagctagctgaattctgaatggaaggaagatgaagaataggtggagatagggcgtttgttctttctcgcttcaccttctgtccctaagtgagcattaaactacattgtaaatatgtcagcaggaacacaagaggtaaagccacacagttccacatgatatcaccattgattttctctctttcagagaattaaagatggcagcaccattgactcagttaaaacgatctgtgtgggggatcatatcgaatgtataaacggagaaaatattgttgggtggcgtcactttgaagtcgctaagaagttaaaggaattgaaaaaggaggagctctttactttgcagttaatagaacccaagaaggcttttggtaagttgagcccgtgtgtggtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgagagagagagagacagacagagagagagagagagagagagagagagagatatggatggacagacagacaaagagaaaccattttcttccccttgtttatctaagaggcaggatactgCTCTGTGGGGCTGTGTGCTTTTAGCATACCATCTTGTCTACTGTACTGTACTAGGGAAGAACACAGAATATTACAGACAGCACTACTTAGAAGGAAAGTAACTCTGAAGGGAAAGGAAAGTTTTCCATTGAAGAACCTTTTTCTAA
This mutation is a 637 bp deletion beginning at Chromosome 3 position 152,127,850 bp and ending after 152,128,486 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
45425 | CAG GGG TTT TAG AAG GAT GTG A | Common | A | |||
45426 | CAA CCC AGT CAC CTA AGA AGG | Wild type Reverse | A | |||
45427 | AGA CAA GAT GGT ATG CTA AAA GCA C | Mutant Reverse | A | |||
45428 | Fluorophore-1 | CAG AGG GAA GAA CAG TGT CTC AC | Quencher-1 | WT Probe | ||
45429 | Fluorophore-2 | ACA GGT ACC TCT GTG GGG C | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
45425 | 0.40 uM |
45426 | 0.40 uM |
45427 | 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 |