Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr3:30800786+30800909 124bp TACCGTTGAGGGAAGAAGGA CTGATAAGCATCTTCACAGCAAG
Mut= 122 bp
Wt= 124 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
CTAATCTCTAAATCGAATGTATTTCTTTGCTAGTATAGCAGTAGGTTGTTCAGAGTGATTCTTTAGTGGTTATAATAACACGAGCATTTTCATTACCGTTGAGGGAAGAAGGAAGTGTTTGAGGTTATTTTTTTGTTGATTATTCAgagattgatggttaacaaatgtttatcttgcttgaataataatatttacttgctgtgaagatgcttatcagtatactcacagttgacataaataacattatagtcaatagacttgatattctgttttattttctgttgtcagaatgttgacatttatagcatgatttcataagacgtaagttcatgaatactaaattttaaataaccaatttcttgtatagcttcaaaagcagtggagtgcctcttggattcaaagtgggccaaggccaagaaaggagaggacgctttatttacaaccagggagtctgtggtcgactactgcaacaggtagcgttccttggtgaaggataacaattagcaataccagttttttccatagtgttgaaactgtttaactaaatgtaatgagctttatatgtaacttggatttttcagtagaaatatctttgttcttaaacttactatatttttagttgtgtatgtatactttgtagttatgttcttttacttgtgtgtatttaccagagctactgaatgGAAGAATCAAGATTTTAAAAAGGATAAACATGTCTACATTGAACAAATTCTGTGTAGGAAATGGTCAGGATTTTTTTTCCAGATCTTTTATAGGATGACGGTTAACTTCATTATCCTTTGGTTTTTAATGGCTTTGCCCTGGGCTATAAGCCTAACTTACTTGCTGTTTT
This mutation is a 537 bp deletion beginning at Chromosome 3 position 30,800,839 bp and ending after 30,801,375 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
41242 | TAC CGT TGA GGG AAG AAG GA | Common | A | |||
41243 | CTG ATA AGC ATC TTC ACA GCA AG | Wild type Reverse | A | |||
41244 | CCT GAC CAT TTC CTA CAC AGA | Mutant Reverse | A | |||
41245 | Fluorophore-1 | TGA TGG TTA ACA AAT GTT TAT CTT GC | Quencher-1 | WT Probe | ||
41246 | Fluorophore-2 | TTG TTG ATT ATT CAG AAG AAT CAA GAT T | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
41242 | 0.40 uM |
41243 | 0.40 uM |
41244 | 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 |