Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr1:191070326+191070441 116bp CCAATGAAGCAGGTCTGG AGGCAAAGACAATGGCACA
Mut= 121 bp
Wt= 116 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletions in lower case):
TTATTTTTTTTTTAAACTGCATCTGTCTGCAGTTACTTCTTCCAGGGTGCTTCATTTCTGTGCTCTGTCCTAGACCTTCTGAGTCCCGATTTACACACTAGTAAGGTTCCTGGGCAATCACTCCTGCTCGAGCTTGAGAACCACTGTCTACAGCCGAGGAAAGAGATGATAAAATAGTTGCCACTGAAGTCTGAGGAAGTCCCCCAAGTCACCATGCCTTTCCTTTCCCTGCCCTGGTGAGTCAGGTCCTAGAGTGGGTGCCCTTCTCACTGCTGTCACAGGTTGGGTTGCACAAAAATAAAGTGAACCCTGTCCATGGTATGAGATGCCTTTTATAGTTTAATTATGGTGGGGAAGGGCAGAGGACTGGCCCAGGGCCCTGCCCATGCGAGGCCCATGCTCTGGCACTGACTGCATCTCTGCCCTCCTCTCTGTCACCTGTCCTATGCTGTTTGGGTCGATCCGTTTGCAAGGCTCAGTGCCGTCAGTTTCCTCTCCCCGAGGTCCTTCTCCCTGAGCGATCTCATCCAGTGCTTTGTCCGCACCTGGATTTCAAGCCTAAGTGCTGTTCTTTATCTCCCACTGCTCCTGCTGCCGTCTTCTGCAGCCTTCGTCTCTGTGGCCTGAGGCACTTATCAGGTGCCTCTTTCTTGGGACTATGCTGCTCGCCTCGTGTTTCCTGGCGCTTGCAGATTAATCAGTGCGgagatgaggggggctccgttaacctgccatgtttgttttctttattctgtgacttatgcttatataatatttctatactaataatggacttctctgaaatagattctgacatcaaagtgcttgaagatgaatttgatgagctcatagtagatgtggcaaccaaacggaggcagtatcccagaaggatccttgagagtgtcatcaaaaccttgaaagcacagcacgcgagtctggtgagacgcttatatctaaatccctacacagtattttaccacagtagagagttagtttacttacctttcatatagtgcaggctggtcttgaacacctgatcttccttctctcactggctggggtcagtcaccatacctatcacatatcagtcttctcagaattattgaaactggagtaaatgcaaatgagccctgaatcataatgccaatgaagcaggtctggcagcatagacctgaggcaaatgctgagGAGATTGGCTGGGTGCCATTGTCCCGAGAAGTGACCACAGCAGGCTTTAGATTGTGCCATTGTCTTTGCCTTTAGTTTTTTAAGTCAGCAGACTTTTAAGTGTGACCTTCCAGTATTGTCTCCTTCAGTAATGCTTTGGGGCATTAAAGGCCTGCACTTGACCTTAGAGTCCTGCTGTGACAGGAAAAAGAAGTCCTTGAGGATGTTCACTTTCTCACACATTCGGCCTTAAAGAGCCCTTCCCACACTGCAGAGCAGAGAAAGCGTTATCTGCCGTTGACTGTGTCATGCCTCCCTCTAACTTACCTCCTTGACCTGCTAGAGAAACTCTGGAACTAGTCAGCTCGAGGAGGGGGGACAGGCCTGGAGAGCATGAGTGTGCCCTGTCACTGACTGTGGCAGGTCCAGCTGCTCCTGTGTGCTTCCGTGAGGAAGACGGAAGACTGCTGTGGTAGTGTAGCCACTTTTTTGAAGTTTCTGTGGAAATAGGAGTTGTGAGTTCCAGATATTTGTCCTGAATGAGCATCCGCCACACTGCGGTCCTCGCCCGGTATTTTAGAGCTGCCTTCCCTGTTTCCTCTGACTGCGATCTTCATAGTGCTGTTTGTTACTCTGCAGCAGAGGGCGGTGCATAAAAGGTATTCAAGCCTAAAGACATTTGCCACCCAGAGTTCAGGAGCTAAAAAGGGTTAAACCTGGAGGCCAGCACTTTTGACTGTGTGTCTGCTCATACCAGCTGAAAGAGAGGCTGAGTCATCTGAGGCATTACGTGTCTAAGGTAAATGTTTTAATTTGTCCTGTGTTTAGAAGCAGTATCACCCTGTTGTACATCCTCTGGACCTGAAATGTGACCCTGACCCAGGTGAGCCAGTGTGA
This mutation is a 483 bp deletion beginning at Chromosome 1 position 191,069,888 bp and ending after 191,070,370 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
48414 | AGG CAA AGA CAA TGG CAC A | Common | A | |||
48415 | CCA ATG AAG CAG GTC TGG | Wild type Forward | A | |||
48416 | ACT ATG CTG CTC GCC TCG T | Mutant Forward | A | |||
48417 | Fluorophore-1 | AGA CCT GAG GCA AAT GCT GA | Quencher-1 | WT Probe | ||
48418 | Fluorophore-2 | ATC AGT GCG GAG ATT GGC T | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
48414 | 0.40 uM |
48415 | 0.40 uM |
48416 | 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 |