Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr2:74859275+74859410 136bp CTTGCTTATCATTTTTAAGGGGAGT AAATGCCTGTGAAGAAAAAGAG
Mut= 124 bp
Wt= 136 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
ATTAAACCATCTTTAAAAATTTGATATTATTCATTTAAGAAAGTAAAGCAATATGTAATATTGCTTACATACACAAATAGAAAATGTAAAATTTTCCTTAAATTTTAAAATTTTATATATTTACTAATGACTTAAGTCTTTTTTTAAGTAAAATATTTTTGTTTGGAAACTTTAAACTTGCTTATCATTTTTAAGGGGAGTACTACTGTATGAACTTTTAAGTTTAGgaaaagaatgaaggaagagttctctttaaatatgcacaagtttttaataaccttattttatttctctttttcttcacaggcatttttgcagatgtgtaatctgcctgtcaaagtggtgtgtagggcaaatgcggaatatatgtctccatctggtaagtgggtgtgttttctattaatacttttgtttgttatttgacattgaggattgaacctaagaccttagctgtgatagcaggtgcattaccactggccttatttctagcacccaacgttaaagtccttagtttgtttgtttgcttgattttttttctgctttgaaggattatttttctattgtttttttaaagtagcagttatgaacttaatcataaagacctatgtgtacttacttgagctctggtccgagGTGTTTTACTGGATTGCTAAGAACATAAGTGATGTCAGAATTCCTTAGTTTTATTTATAGGGCTTTCCTTACCAGGAAACAAATTTTAATGCTGATGAATCTGGTTTTTGTATGGTTTTGTTTGTTTGTTTTTTTATTTACATTCCAGCCATTGCACCCCTCCCCCCTTCCCTGTCGCCCCTCCCACAGTTCCCCATCCCATTTCTCCTCCACTTGTCTCTGAGAGGGTGCTCCGCTCCCACCAAACCAGCTTCCCTGAGGCTTTGTAGTACAGCTTGAGGTCTGGGATGGTGATTCCCTGG
This mutation is a 406 bp deletion beginning at Chromosome 2 position 74,859,326 bp and ending after 74,859,731 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
48230 | CTT GCT TAT CAT TTT TAA GGG GAG T | Common | A | |||
48231 | AAA TGC CTG TGA AGA AAA AGA G | Wild type Reverse | A | |||
48232 | GGT AAG GAA AGC CCT ATA AAT AAA AC | Mutant Reverse | A | |||
48233 | Fluorophore-1 | AGG AAG AGT TCT CTT TAA ATA TGC ACA A | Quencher-1 | WT Probe | ||
48234 | Fluorophore-2 | AAG TTT AGG TGT TTT ACT GGA TTG CTA A | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
48230 | 0.40 uM |
48231 | 0.40 uM |
48232 | 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 |