Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr5:137562951+137563119 169bp TGTGAAGTGTGTGTGTATAGGAATG CCCGAGTGTTCCACTGTGA
Mut = 190 bp
Wt = 169 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
CGTATTTGTTCTGAGTATGGACAAATGTGTGTGTGCACACACACATACATACTAACTGACATgtgggctgggcctccatctggactcttctcctgtgctcttctcacagtggaacactcgggccaagcgggagaagctgacggagctgatgttcgagcagtacaacattcctgccttcttcttatgcaagacggccgtgctcacagcgtatcctacatctcgggcagtcctgctgggtggaaggctagagtggggtgtgtgttgagctgggtggggctgggcctgattccctggagatcagtatctcgggtaggaagaatcctcttagcacctcactagtgcagaaggccccataGGCTGGAGGCTTGGGCTAGGGGCGGAGGTTGCAGATCTGTGGCCCTTCCCTATTTTACTTAGTTTATGGGTCAGTCTCAGCAGGCAGA
This mutation is a 293 bp deletion beginning at Chromosome 5 position 137,563,060 bp and ending after 137,563,352 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
49911 | TGT GAA GTG TGT GTG TAT AGG AAT G | Common | A | |||
49912 | CCC GAG TGT TCC ACT GTG A | Wild type Reverse | A | |||
49913 | GCT GAG ACT GAC CCA TAA ACT AAG | Mutant Reverse | A | |||
49914 | Fluorophore-1 | CTG GGC CTC CAT CTG GAC TCT | Quencher-1 | WT Probe | ||
49915 | Fluorophore-2 | CTG ACA TGG CTG GAG GCT TGG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
49911 | 0.40 uM |
49912 | 0.40 uM |
49913 | 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 |