Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr19:5091599+5091696 98bp GAGGTGTTGGCTCTGCTTCT CCCACATATTTGTAACCGCTGTAAG
Mut = 110 bp
Wt = 98 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
GAAATCAGGCTGGTACCTGTCTAGGATGAGCAGAGGTCTTGACCGTGGTGTTAATTCTGGAGATATTGCAGGGAGCTGATGAGTTTCCTAATGGGAGGGAAGGTGATGGCTATGGTCCTGGGAGGCCCAGTAGTAGTCAGCTGGAAGGGGCAGTCGAGAGGcgcaccggggacaggacattggagagtgaagttgggatatgcagggagggaaggatgggagaggctggagcactagtggacttgaatgcctaatgaggggctctgggtgaggaagccagaggaactacttcctctcttctcttctcttggcagccatggccttcatcacatacgtgctgctggctggaatggcgctaggcattcagcaaaggttagtattctcttctccctcctggcttatgtcccttcctctgtagggaatctgtgccaggtcctagctcacccaccttgggcctaccctgacctaccttctcccatggctgcaggttctccccagaggtgttgggcctgtgtgcaagcacagcactggtgtgggtgttcatggaggtgttggctctgcttctgggcctctacctggccaccgtacgcagtgaactgagtaccttccacctcttggcttacagcggttacaaatatgtggggtgagtgcctgcaggctgagaggagcctgaagggtctcaggctacaggttttgggcctatgtagcaacagccttcactgtgatgctagagacagttctactccgtctcaaccgccagctctacgctgtgagttggtggctgtttctgacagtctccccttggagCCAGTGACTTTAAAGAGACTGGGGATTTGTTAGTTACCTTGGGGAGCCCAGAGAAGGGAAGGGGAGTTTAGGCCCTGGGACTGAGTCCTGAACCTTGCAGGTCTATTTGGAGTCTGGTTCTTTGTA
This mutation is a 646 bp deletion beginning at Chromosome 19 position 5,091,215 bp and ending after 5,091,860 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
50911 | GAG GTG TTG GCT CTG CTT CT | Wild type Forward | A | |||
50912 | CCC ACA TAT TTG TAA CCG CTG TAA G | Wild type Reverse | A | |||
50913 | GGG AGG CCC AGT AGT AGT CA | Mutant Forward | A | |||
50914 | AAC TCC CCT TCC CTT CTC TG | Mutant Reverse | A | |||
50915 | Fluorophore-1 | CAC CGT ACG CAG TGA ACT GAG | Quencher-1 | WT Probe | ||
50916 | Fluorophore-2 | AGA GGC CAG TGA CTT TAA AGA GAC TG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
50911 | 0.40 uM |
50912 | 0.40 uM |
50913 | 0.40 uM |
50914 | 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 |