Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr3:54807127-54807276 150bp TACAAGAAGAAGAAGAGCGACCAG AGACCCTGAGTGTGTACCAAGT
Mut = 161 bp
Wt = 150 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
GCTGGCCTCGAACTCAGAAATTTGCCTGCCTCTGCCTCCCGAGTGCTGGGATTAAAGGCATGCGCCACCACGACCGGCTGTCAGCCGGGTTCTTGGTCAGCCTGCACCTAGGAATCGCTTGCGGATGGCCGCCGCGCAGGCGCAGTCGGTTCTGTCTCGCACCTGCTCAACCCCGCCCCTCTCGCCGTTCCCAAGCAGAGGCTCGCGGACCGCTACGGCGCACGCGCAGTGCAGTGCTGGCGGTTCGGCTCCGGGACGTGCGGCGGGCTGCCGCTTGTCGAGTACGGTTGTGTTTCTCAAGAGCTGACGGGCCGCGGTGGCGGACCCGGGCTCGGGCGGCAGCACGGAGGCGCAGGCTGTGCccgagggctcggggccgagcaccgccagtccccggacggcccctccggtgacggtgctggtgatgcggcaggacgaggccgaggcggacggcgctctgaggccgggcctggcggggagtgaggcggcggcagacgccgaggacgaggcgggggacgacgacgccgacctgctggacacatcggacccggcgggcggcggcgagagtgcggccagcccggaggagctggaggacgaggacgcggagggtggcggcgcagcgcgcaggcgaggctccaagacctgcacgtacgagggctgccgcgagaccaccagccaggtggccaagcagcgcaagccctggatgtgcaagaagcaccgcaacaagatgtacaaggacaagtacaagaagaagaagagcgaccaggcactgggctctggcggcccgtccgcggccagcacaggcaacgtcaaacTGGAGGTATGCTTGCTCCTCGGGGCCCCGGGGCCAGGGCAGATGGGTGCCAACCTACTTGGTACACACTCAGGGTCTCCCCAAGGGCTCATCCTCTGCTCCAGCTGACATGGGCACTTTGGAGGTGATCTACACCTCTCCTTAGTAAACCCTTCCCAGTGTCTACCCCGAGGCATCCGGCACATTGACACACCCTGTTGGTTTTCTGTAACTTTGGTGACTTTGTTTTTTTTTCTTAGACTCCCTGGCCCCTGAGAAA
This mutation is a 453 bp deletion beginning at Chromosome 3 position 54,807,204 bp and ending after 54,807,656 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
49103 | AGA CCC TGA GTG TGT ACC AAG T | Common | A | |||
49104 | TAC AAG AAG AAG AAG AGC GAC CAG | Wild type Forward | A | |||
49105 | CGA GTA CGG TTG TGT TTC TCA | Mutant Forward | A | |||
49106 | Fluorophore-1 | CTC TGG CGG CCC GTC | Quencher-1 | WT Probe | ||
50046 | Fluorophore-2 | CGC AGG CTG TGC TGG AG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
49103 | 0.40 uM |
49104 | 0.40 uM |
49105 | 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 |