Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr17:87290576+87290663 88bp GCTATCCACTGGGTTCCAGA GTTGCCTCTCCCAGGTCTCA
Mut= 86 bp
Wt= 88 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
ATGTGAGAGAATTGAAAATGACGCAGGAAACCGTACATGCCACATTCACTCTTTGGTTTCTAGGTCGTCCTGAGCAACATTAAAGTCCCTTAAGAGGAGACAAATTGACTACACAATCTCACAAACTAGGAGACTGTGAAACGGTCGATGAAGCCCTCACAATACGGTTCTCCTCTTCCgtggtttgagtgggtgggtgctttttgttttccaatgggttcttgctctgtcacccggactgagtgagaactctcttccgtcctcagagacgatttaggtttatggagaatgcacctttatcgtctcagacttgagccattcgtgcgctgggcagggatggctcggggctcaggatatctacagcgcatgttcaaggagtcaggaatccttgtcctgtttttccagatgactttgtgaaattgctgctagcggaggccctcctggagcagtgtctgaaggataaccacgacaaaataaaaaactccatccctttgctggagaagactgatcacaggctgaatgaagccaaggaccacctgagcagcctcctcaacaatgggaaactgccggtgagccttcaggttgtctcactcagaggtcagggcagagcggggatctctaggcagcaggtggattcttgtgtatcccaagtagctccagtggatatggagaagaggcgggaaaggcagtcccaagataaaaatcagaatcctacaatctgagtgattcctgaagtcccagctagctcagctctgtggagatcagtgacgtgaagccgctgccctagcttctgctatccactgggttccagaactgcagcatctcattaaggcttcctaggtacTTTTTAGCCCCATGTCTGAGACCTGGGAGAGGCAACCACATAGCCAAATTGGTTTCACCTGCTGTGGTGTCTCAAGCTCTTGAGATGCCCAAAGAACTGGAGTGGGAATAAAATAATGACCAAGGAACTTGGGCCAGTGGAAGGACAGCTTCTAGGGGTTTCGCGATCAGAGT
This mutation is a 665 bp deletion beginning at Chromosome 17 position 87289963 bp and ending after 87290627 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
39513 | GCT ATC CAC TGG GTT CCA GA | Wild type Forward | A | |||
39514 | GTT GCC TCT CCC AGG TCT CA | Common | A | |||
39515 | GAG ACT GTG AAA CGG TCG ATG | Mutant Forward | A | |||
39516 | Fluorophore-1 | TGC AGC ATC TCA TTA AGG CTT | Quencher-1 | WT Probe | ||
39517 | Fluorophore-2 | TCT TCC TTT TTA GCC CCA TGT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
39513 | 0.40 uM |
39514 | 0.40 uM |
39515 | 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 |