Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr3:89243496-89243589 94bp CCCAAGGTGAGCCAGTTCT TAGAACCCCACATGCCAGTC
Mut = 101 bp
Wt = 94 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
GGCGCAATGAAATTAATTCCACGTTTTAATTGATCAGATGTCACCCACCCCAGCCAGCTgtaattctcacctgtgcctcctgggtccatctacaggctttgggacataccctgggcggaagcgtggtgccttgcacccccagacgatcctattcccgtgcccagcctgccaaggcgatgtggagctgggggagaggggcctttccgggcttttccgaaacctgaccctagagcgagtggtggagaggtaccgccagagtgtgagtgtgggaggagccatcctgtgccagttgtgcaagcccccaccactagaggccaccaagggctgcacagagtgtcgagctaccttctgtaacgaatgcttcaagctcttccacccctggggcactcagaaggcgcagcatgagcccacgctacccactctttcattccgccccaaggtgagccagttcttgcgggggtctgaagtgggaggggctacagtggtgctgaaccAGAGGTTGGAGATGACTGGCATGTGGGGTTCTAAAGAGGGCTAGGTAAGGTTTTGTGTGTGTGTGTGTGTGTGTGTGTG
This mutation is a 435 bp deletion beginning at Chromosome 3 position 89,243,529 bp and ending after 89,243,963 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
53135 | TAG AAC CCC ACA TGC CAG TC | Common | A | |||
53136 | CCC AAG GTG AGC CAG TTC T | Wild type Forward | A | |||
53137 | CTG ATC TCT GGC GCA ATG | Mutant Forward | A | |||
53138 | Fluorophore-1 | CTG AAG TGG GAG GGG CTA CA | Quencher-1 | WT Probe | ||
53139 | Fluorophore-2 | CCA CCC CAG CCA GCT AGA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
53135 | 0.40 uM |
53136 | 0.40 uM |
53137 | 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 |