Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr1:54550979-54551108 130bp TGACAAAGCGTTATCCAGCA TGCTTATAGCTTCCAGCATTACC
Mut = 142 bp
Wt = 130 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
CAGTGACAGATAGCCATTGTCAGGGATTATTTTAACTGATTGGTTAGAATTTCAACCATACGTATTTTATGCTTTTATGAAGGTTATACTCTTCAAGATCCATATAATTTAAGAATAATCCCctgcgtgaaaaacgataacttgttttgtgcattgggtatggtttatagcattcataatatttgtatactgtaccattttaaagttctttatgtttgcattgagtctgagttgtattaataaaagtgattaaaatggacccatttatttatttatttatttatttattttctgtagaaaatagagctcccaaagaaactgacaaagcgttatccagcatatgagttatatctttacggagaaggatcgtacgctgaagaacacaaaattctccctttgacaggaattcctgttctctttcttcctggtaatgctggaagctataagcaaggtaggtagtggaagaaagacgttaaagctgtgagactcagctccagacttgcaatcatgtgcgtaatgccagagtcaagtgctgtaagtgaaaagcttactctgtcaggtactgtacatgccacttcccaaaagaaagcaaacatttaagatagaaggaaaagaagagtcgaacactacttcgcagagagtgagaagaatgagatggtgtaggtgggcgaatagattatacaggcattcagaaatcagatggcatagggtagggggaatagattatagaggtactcAGAAGTAGAGGAAAGGTTAATGTTGGAAATGAAGTTTCTGAGAATGGAATCTGCAACATTCAGCGGGGAGAGAAATCTTAAATTATGTTGAACAAGGAAGAGATC
This mutation is a 615 bp deletion beginning at Chromosome 1 position 54,550,691 bp and ending after 54,551,305 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
51391 | TGA CAA AGC GTT ATC CAG CA | Wild type Forward | A | |||
51392 | TGC TTA TAG CTT CCA GCA TTA CC | Wild type Reverse | A | |||
51393 | AGC CAT TGT CAG GGA TTA TTT TAA C | Mutant Forward | A | |||
51394 | CAT TTC CAA CAT TAA CCT TTC C | Mutant Reverse | A | |||
51395 | Fluorophore-1 | ACA AAA TTC TCC CTT TGA CAG GA | Quencher-1 | WT Probe | ||
51396 | Fluorophore-2 | ACC ATA CGT ATT TTA TGC TTT TAT GAA GG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
51391 | 0.40 uM |
51392 | 0.40 uM |
51393 | 0.40 uM |
51394 | 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 |