Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr12:85272294-85272409 116bp ACCTTATGCTTACATTGCTTCC TTTCAGACCTCGTTAAACTACCC
Mut = 122 bp
Wt = 116 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
TAGTTCCGGTGGTTTGATTCCAGGCCAGGATGCTGGCCCGATTCTGTCTccctttgttccgtgcagtctcaccgcacctctctttctctcttttcttcccttaggctgagggtaaaaagctaggtttggtgggctgggttcagaacaccgaccggggcaccgtgcaagggcaactgcagggccccgtctccaaggtgcgcttcatgcagcagtggctggagaccagaggaagtcccaagtcgcacattgacagagcaaacttcaacaatgagaaagtcatcgcaaacttggattattcagacttccaaattgtaaaataatgaaacgaatcttaatattttttcaaaataatctcactcctttttttaaatcgctagattaaaaaaaaaatagaactattctgtgctcagtattagaatttgttagtaagttattttggttgcatgttggaaaagttaccacgtattacaagtatgatgaaatacaaatgtgtataattctaaccaataaaaacacattagaaccttatgcttacattgcttccccccctcccttttaatccttagtgaactttgggcaacatgattaattcgttattgtGAGAGGAAAATTTTGGGGTAGTTTAACGAGGTCTGAAAGAGAAGTAACTTTGCCATTTCTTTACATTGTTTAC
This mutation is a 551 bp deletion beginning at Chromosome 12 position 85,272,332 bp and ending after 85,272,882 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
53061 | TTT CAG ACC TCG TTA AAC TAC CC | Common | A | |||
53062 | ACC TTA TGC TTA CAT TGC TTC C | Wild type Forward | A | |||
53063 | GGT GTC TGT TTT GAT TTT TCT GGA C | Mutant Forward | A | |||
53064 | Fluorophore-1 | CTT TGG GCA ACA TGA TTA ATT CG | Quencher-1 | WT Probe | ||
53942 | Fluorophore-2 | ATT CCA GGC CAG GAT GCT G | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
53061 | 0.40 uM |
53062 | 0.40 uM |
53063 | 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 |