Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr10:13182741-13182863 123bp TACGTATGTGTGGGGCTTGC TTGTGCACTGAAACAGATGTC
Mut= 121 bp
Wt= 123 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
AAGCATAGATTCATAACAGAAGTATTTTTACTAGGAAACGTTGAAGAAGGATGTTTTGTTTTGGTTTTTTTATTCTATCTTCCTAAACTGGACTTAAGTGTTTCAATCCTTCTGTTCTCTGCATACTTACTTAGATGTGATTGTATACAGTAAACGGAGTGGCTTTCTTGCCCCCCAGAGGGATACAGAACTCAGGACTTCTGTTTTGTTTTCTTTATAGCAGtggagcccaggcaggtgtccaactaacatcaaagctgaggttgaccttgaactcctgggcgctggaattaaagatacgtgttttactcagtttctcttttccttgtaggacctcgactggattttgatccagacatcgttgcagctcttgatgatgattttgactttgatgacccagaaaacctccttgaagatgactttattcttcaggccaacaagccgacaggaggagagaggatggatacgtatgtgtggggcttgccatactggtgtgtgtgtgggggggggggggtgggggtaaccatgttctagtagGAAGACAAGTTTGAGAGCACGTAGTCTAAGACATCTGTTTCAGTGCACAAATAGTTAGATTTGAACATATATTTCTGTAGTATCTGAGATCCCAGAACCTGGTGGCTGATGGCTTTTCCCACCACGCCAGGCTAGTGTAGCGCCATCTTGAGTCCCGTTCCCTCTGGAGCCTCCAGCAGTTGCTCCAGAT
This mutation is a 314 bp deletion beginning at Chromosome 10 position 13,182,791 bp and ending after 13,183,104 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
46877 | TAC GTA TGT GTG GGG CTT GC | Wild type Forward | A | |||
46878 | TTG TGC ACT GAA ACA GAT GTC | Common | A | |||
46879 | AAC GGA GTG GCT TTC TTG C | Mutant Forward | A | |||
46880 | Fluorophore-1 | TGG GGG TAA CCA TGT TCT AGT AG | Quencher-1 | WT Probe | ||
46881 | Fluorophore-2 | TGT TTT CTT TAT AGC AGG AAG ACA AG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
46877 | 0.40 uM |
46878 | 0.40 uM |
46879 | 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 |