Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr7:65647244+65647363 120bp CAAGGGTTGTCTATTAAGAAGTGC TCCCCTCTTACCTAGTGTCTCTG
Mut = 119 bp
Wt = 120 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
ACCAAGGGTTGTCTATTAAGAAGTGCTACACTGTATATATAATATTGCATAAATATACAgtcagtggtgggagagggatacgtccatgagtgtagtattcagagacactaggtaagaggggaagggctgctggaggggacatcaagagagggggagcatgcggagaggtgaagtaggacccgctgagccaatagctccgtgggctgagtccttaacggtatgctaatgactttcaggtgaagcaaccgaattttataaaagagagattgcagctttttgaaacattgaagacagatcatcaactgttacctgctactcaggaaaagaagaacacaaacaacgtgatttcagtgagggtggctggtgggaagacagtgcaaggagaaaggtggaaaacaacaccttatcaagtggccgctggaattaggtaaggcccgcccccttgccgacgccccgcccccgccagacgcatgcgcacaagcatacatacctgcccacagactccttgttcgtctattttcctccctacctGTTTGTTTGAAGCACATATACTCCAGCATTAAGTAATTGTGCCAGAGCCCGTTGAAAGGTTTTATTGACACTAAAATAT
This mutation is a 472 bp deletion beginning at Chromosome 7 position 65,647,301 bp and ending after 65,647,772 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
51247 | CAA GGG TTG TCT ATT AAG AAG TGC | Common | A | |||
51248 | TCC CCT CTT ACC TAG TGT CTC TG | Wild type Reverse | A | |||
51249 | AAA CCT TTC AAC GGG CTC TG | Mutant Reverse | A | |||
51250 | Fluorophore-1 | AGT GGT GGG AGA GGG ATA CGT | Quencher-1 | WT Probe | ||
51251 | Fluorophore-2 | CAG TTT GTT TGA AGC ACA TAT ACT CCA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
51247 | 0.40 uM |
51248 | 0.40 uM |
51249 | 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 |