Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr9:107847034-107847158 125bp TCCAGAAGTTTCCTGTTGGTG CAGGCACTCAGAAGAACTGGA
Mutant= 92 bp
Wild Type = 125 bp
Wt Sequence: GAACCTCATTCTTAACCCAGCTCTTATTCCTATGTATATTGTATTCAGTTTCTGTTCCTTTCTCTCTGTGGTGTGCTTCCCTCTGTTCATGGTGACATAAAGCCCGtgtgattcttggccagctgggattataggtttgtcattatgcctaatttcatttagcacattttgaattcttacagtgtatatggtttaattttgtcttcataaaaatttgtagatttagttatataaagtaaaaaccagtagggaaataaaaaattagcacttaacatgctttgtataattagtgtaaatgtcatatttttgcagattcttaatgcctttcgaacttctgatggcatacctgtaaaggacttgcagttgaaggagtataacacaggtgagttttttagatgtgcatagactcttggtttaggaagggtcttggagatatgtctgcatcatatgctactcaaaaatttgttttttaagaaatcacaagtatgatttccagaagtttcctgttggtgccttcaaataacaagcagccttagtcttaactgtgattctttgtatggctctttgtccacacATGGGCGATGAGGATGCGTGTTCCAGTTCTTCTGAGTGCCTGTGAGATATAAAGTGAGTGTCATAGCATCTATCCCTGTTTTATATGATTAAATTATTAAACCTAGTATTTATGTTCTTCACAATGGTGGTATGTTTACATTTGAGCCTA
Deleted Sequence: tgtgattcttggccagctgggattataggtttgtcattatgcctaatttcatttagcacattttgaattcttacagtgtatatggtttaattttgtcttcataaaaatttgtagatttagttatataaagtaaaaaccagtagggaaataaaaaattagcacttaacatgctttgtataattagtgtaaatgtcatatttttgcagattcttaatgcctttcgaacttctgatggcatacctgtaaaggacttgcagttgaaggagtataacacaggtgagttttttagatgtgcatagactcttggtttaggaagggtcttggagatatgtctgcatcatatgctactcaaaaatttgttttttaagaaatcacaagtatgatttccagaagtttcctgttggtgccttcaaataacaagcagccttagtcttaactgtgattctttgtatggctctttgtccacac
468 bp deletion beginning at Chromosome 9 negative strand position 107,847,543 bp and ending after 107,847,076 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
37478 | TCC AGA AGT TTC CTG TTG GTG | Wild type Forward | A | |||
37479 | CAG GCA CTC AGA AGA ACT GGA | Common | A | |||
37480 | CCT TTC TCT CTG TGG TGT GC | Mutant Forward | A | |||
37481 | Fluorophore-1 | CAA GCA GCC TTA GTC TTA ACT GTG | Quencher-1 | WT Probe | ||
37482 | Fluorophore-2 | TCT GTT CAT GGT GAC ATA AAG CC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
37478 | 0.40 uM |
37479 | 0.40 uM |
37480 | 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 |