Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr12:104266648+104266750 103bp AAGATGCAGCAGGTGGAAAC CCTAACCAGGTCTGGGTCCT
Mut = 107 bp
Wt = 103 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
ATTCAGAGTTCTGTGTCTTCCAAGGGGTTTCTAATCATTATAAAACTACTAAGGGACACTGTGCTGTTGAAGGTATATTGACAGctgtcaggactcagcagtgggtgccatatagcatggaggtgcgctctctgtgctagtgttacaatctgatgatttgctccacatttaggcaaatggaagatgccctttgacccccgtgatacatttaattccaagttttacttggatgagaagaggtctgtgaaggtgccaatgatgaaaatagaggaactaactacaccctacttccgggatgatgagctgtcctgctctgtggtagagctgaagtacacaggaaatgccagtgccctgttcattctccctgaccagggcaagatgcagcaggtggaaaccagcttgcatccagagaccctgaggaagtggaagaactctctgaaacccaggtgcatacccacaggacccagacctggttaggattcccttagtgctgttgtcctgatatctagtatcccagtacctcagagtctcacttacagcctgtgtaattgttaaagtgtcagcaggggacaggtgaagttgaattgagttccctccactgtctcaggtgtttcaggcctgattttactgtggtttttgttcttcacaatatgtgacaatctcacacagtgagatgaggtttttccccCTCTCTAATAGCCTGTAAATTATATGGAAATGATTGAGAGTTGTAAGCCACAGGGATTGTGATTAACAGGAGAT
This mutation is a 604 bp deletion beginning at Chromosome 12 position 104,266,357 bp and ending after 104,266,960 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
50127 | AAG ATG CAG CAG GTG GAA AC | Wild type Forward | A | |||
50128 | CCT AAC CAG GTC TGG GTC CT | Wild type Reverse | A | |||
50129 | AAA ACT ACT AAG GGA CAC TGT GCT G | Mutant Forward | A | |||
50130 | AAT CAC AAT CCC TGT GGC TTA C | Mutant Reverse | A | |||
50131 | Fluorophore-1 | CCA GAG ACC CTG AGG AAG TGG | Quencher-1 | WT Probe | ||
50132 | Fluorophore-2 | TGA AGG TAT ATT GAC AGC TCT CTA ATA GCC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
50127 | 0.40 uM |
50128 | 0.40 uM |
50129 | 0.40 uM |
50130 | 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 |