Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 75 bp
Wild Type = 114 bp
>chr8:109422442+109422555 114bp GGTTCGGGCAATGAACAG CCCTTGTGCTCAGAGAAAATC
Wt Sequence (deleted regions are in lower case)
TTTTTCCCAGGTCATGAGTCTCGGGGTACCGAAGCCCTGTGTTGGGCAGGAGGACAGCGGCTCTTTAGTGCTGGACTGAACGGAGAAATCCTTGAGTATGACCTACAGGCACTAAACATCAAGTACACTTTGGATGCCTTTGGAGGACCCATCTGGAGTATGACCGCCAGCCCCAGTGGCTCTCAACTTTTGGTCAGTAAGCTAGTATTGATAATACAAAATACAGTAGTCTTGATGGTCAGTCCCttccccgtctcacaagctgttttggtttgttttgaTTTTAGGCAAGTGATAGCTTTAGAGAAGAAACGCTTGTgcatgggggtattagttctgtataaaggctcatttagtctgttttctactcgtctctaatcaggttggttgtgaagatggctccgtgaaactatttgaagtcaccccagaaaaaatccagtttgcaagaaattttgatcggcaaaaaagtaaggcttgttttgggttcgggcaatgaacagcttcaacaagaggttgtctgtttgtgtcaccaaagggacaggatattttcctgtgGCTATAGGATATGCTGTGTAGATTTTCTCTGAGCACAAGGGGAGTTCTAGAAGGCATCCCATTAGGTATCCCATTAGGTAGCATAGTTGTGAAATTCAAAGTAGAAAGCTAGTGTTCCTGTGAAAATGAGTGTATTGTTTATCACCTGCATTTTAGTCCAGGTTTATGTTGTGATAAACT
236 bp deletion beginning at Chromosome 8 positive strand position 106,898,379 bp, and ending at 106,898,614 bp (GRCm38/mm10)
In addition there is an additional 35 bp deletion (ttccccgtctcacaagctgttttggtttgttttga) 38 bp before the 236 bp deletion
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Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
35294 | CCC TTG TGC TCA GAG AAA ATC | Common | A | |||
35295 | GGT TCG GGC AAT GAA CAG | Wild type Forward | A | |||
35296 | Fluorophore-1 | ACG CTT GTG CTA TAG GAT ATG CT | Quencher-1 | MUT Probe | ||
35297 | Fluorophore-2 | CAA GAG GTT GTC TGT TTG TGT CA | Quencher-2 | WT Probe | ||
37099 | GGC AAG TGA TAG CTT TAG AGA AGA | Mutant Forward | A |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
35294 | 0.40 uM |
35295 | 0.40 uM |
37099 | 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 |