Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr2:27462409-27462503 95bp GATGCCTCAGGAGGAAGTTG GCCTTCTTTTACCTGCATTTTG
Mut = 100 bp
Wt = 95 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
GCGTGTGCCACCACGCCTGGCGTAAAGACTGTTTTTATGCTTTGCATTCTTCTCAGATTTTCCACGTTGAAAAATAATCTTGTcctccctccatgacaaatctcttaacaaatatagtgctttcttgaactataacctacagtgttgttttttgcaagtgcacaactcctttcccagtgagtgacctgggttctctgtcttcagcccacagatgatatagtgctaatggcccaggccttagagaaaatctttctgcagaaagtggcccagatgcctcaggaggaagttgaactattgccccctgctccaaagggcaaaggccggaagccagctgcaggagcccaaaatgcaggtaaaagaaggcaggcagggagggagggagcacaggctttccctgctgaggctgtgggactcctttgatccctctaagtttttACTGTTGCCTGACAGGTAGGACAGGTGTCATGAGCTTGGCTGGTGATGGATCCTTAGCTG
This mutation is a 352 bp deletion beginning at Chromosome 2 position 27,462,338 bp and ending after 27,462,689 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
51878 | GAT GCC TCA GGA GGA AGT TG | Wild type Forward | A | |||
51879 | GCC TTC TTT TAC CTG CAT TTT G | Wild type Reverse | A | |||
51880 | GCA TTC TTC TCA GAT TTT CCA C | Mutant Forward | A | |||
51881 | CAG CTA AGG ATC CAT CAC CA | Mutant Reverse | A | |||
51882 | Fluorophore-1 | CCC CCT GCT CCA AAG G | Quencher-1 | WT Probe | ||
51883 | Fluorophore-2 | CTT GTA CTG TTG CCT GAC AGG TAG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
51878 | 0.40 uM |
51879 | 0.40 uM |
51880 | 0.40 uM |
51881 | 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 |