Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr3:32715324+32715437 114bp CTCTTTGCCAATATGGCTCTG CTCTCTTCGCCCTGGTGTTC
Mut = 106 bp
Wt = 114 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
CCTGTTGAATTATAGAGTTTAATTTATGATTTTTTAAAAAGAGGAACTTAACTGACTGACTTTCTTTCTTTCTCTTTCTCTTTGCCAATATGGCTCTGTAATGTATGAAATGAGTCACTGCTTTAATTActgttcagtggataacccgggtccttcctcccttctctagtggaacaccagggcgaagagagagaaactgacagagttgatgtttgagcactacagcatccctgcattcttcctttgcaaaactgcagttttgacggcgtatccttaaactataagacagcttttctcagtgtgtgtaagacattagaggaagaagtgttttctagctttttacctgagttttttaggcatttccctactccccaccccgtttgaatcattgtatgcagtgttgggtttgggtttcataaagacagtctccttcaggtgtatcccttctccttcaagaagtatatccaatatctcCTTCAAATCGGCGACCATATTCACCCACTTCCTCTGCTTAGATTCATCTGCCCCTCTTTGCTGCCTCCCACTCTGCCTTGCAGCTTTGTGGTGG
This mutation is a 345 bp deletion beginning at Chromosome 3 position 32,715,376 bp and ending after 32,715,720 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
50632 | CTC TTT GCC AAT ATG GCT CTG | Common | A | |||
50633 | CTC TCT TCG CCC TGG TGT TC | Wild type Reverse | A | |||
50634 | GGG GCA GAT GAA TCT AAG CAG | Mutant Reverse | A | |||
50635 | Fluorophore-1 | AGT GGA TAA CCC GGG TCC TTC | Quencher-1 | WT Probe | ||
50636 | Fluorophore-2 | TAA TTA CTT CAA ATC GGC GAC CA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
50632 | 0.40 uM |
50633 | 0.40 uM |
50634 | 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 |