Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr1:163704109-163704204 96bp ATAGGAAGGGTCAGCAAGCA GTAGCTCTGGGCTTCAAGGA
Mut= 98 bp
Wt= 96 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TCTTCATAGAGTTCTTCGTGGTTCACTTCTTGGAGAAAATTACCTTGAATTGGAAGCCAAGATAATAGTTTGTGATTCTTGTTAAAGAGATAAATTTGGGAAAACCAAGCAGAACTATAAGTATTTGGCTTTGGGGGGGGAGGGGAATAATGAAATGTTCTCTCTATTGATGCAGTAGTAGATActtacctataaacacgtgtacaacaaatatgttgagtcacacacataaatgaaacattttatagtctaaaattgagccaaataaacttattttaaagaaataattgtaatgccgagactcgaggtgagtctctgtccttgaagtggcccacaaggtatttttctgcaggggccagggagagcgggaactggctgcgccttggactgcggttctggaataggaagggtcagcaagcacgtcttgttgcctgttttcagcagtgttgaattggtggacatgatggaatccttcctccttgaagcccagagctacttgcaggttaacgaggacaaagttgaaagttaccactgctacagcctgcaggagttcacgccccacttagggagatatgatgtcatctggattcagtgggtctcaggtcagtgttgcacctcatagcttccgcccttccccatttcctccttaggaaaggtgacacttgggactttcaacacgttttgctagctcaggctgtcatgtgaactcccaacaactctgggccctgaagttctcttaacattgctgcacaaacttggcaattcttacccctagggataGAGCTGGAGCCTGAAACAGCCTACATCTTTGCAGAAATGTAAATACTTGAAAACATTGGACCCATAAATGGTTAATCCTCATTATTTATTGGTAGAACTAAATGTTCTGAGACTTAAATCCTTAGGAAATGAAACAGCAATGAATTATATTCTCTCTCTCTTCCTCTCCCTCTTCCTTT
This mutation is a 596 bp deletion beginning at Chromosome 1 position 163,703,825 bp and ending after 163,704,420 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
46261 | ATA GGA AGG GTC AGC AAG CA | Wild type Forward | A | |||
46262 | GTA GCT CTG GGC TTC AAG GA | Wild type Reverse | A | |||
46263 | GAA ATG TTC TCT CTA TTG ATG CAG T | Mutant Forward | A | |||
46264 | ATG GGT CCA ATG TTT TCA AG | Mutant Reverse | A | |||
46265 | Fluorophore-1 | CTT GTT GCC TGT TTT CAG CA | Quencher-1 | WT Probe | ||
46266 | Fluorophore-2 | ATA GAG CTG GAG CCT GAA ACA G | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
46261 | 0.40 uM |
46262 | 0.40 uM |
46263 | 0.40 uM |
46264 | 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 |