Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 105 bp
Wild Type = 105 bp
>chr10:94294873-94294977 105bp TTGCAACTGCTAGGCTCAG TGGCTCATGAAGCAAATTCA
Sequence (deleted region in lower case)
AACAGGGTGACTCACTTCAAATGACGGCCGACAATGAATGATATACAATTAAGCGTGTGGGCCCCAGGGGCAGACTGCATGGACAGAGTCCTGGCTTTGTGATTTTGGGCAATTGAATCTTCTTGTGCCTCAGATTCTTCATTTGCAAACCAGGAACCCTAATGCTGCCTATCCTTGGAGACTAGGATGGGAAAGGAAGCCTGCTACATATGACTCAGCAGCAGAATTGTCACCCTGATGgtcatgggagtctgcttgcttggactaaacctttttatttctctccttattccaggactgcttgtgcccctcccccacacagcccagtggccaggattcctgcctaactatatcccaagaattgagcctaggactctctttggtctgctctggctcataaaagccaagacgaggtgaggcactcctttctgtttctgcttctccgttcactcaccaattcacaaatgattttctgcatgtgtgtttgtgtaatagagaactgtccctttaaatgtgtgtgtcacagatcttgtctcctgctgggattgcagtggcctgaaggcagggaacaaatactgccacaaaaggccttaggctttgtagtacacatggtctcagttgcaactgctaggctcagctgacaaagcatgaggtagccttagacaagatgagagatttcgtttacaaagagcaggcCACTGGCTGAATTTGCTTCATGAGCCATAGTTTCCTAACTTCCAAGCTAAAGCATATTTTAAAAAATTGTTTTTCATCTCTCATTCCCTGTCCGGCTTTTTAGAAATGATTGGGATTATGCTGTGTCTGTTTGCTTATTTTTAAAGCATAAGCAGGAGGCTTTTTGTGGATCTGTATTCGACTTTACTATATTGGCTATGCACGTTCCTCTAGCCTTTCTCCTGTGTTTGTG
456 bp deletion beginning at Chromosome 10 negative strand position 94,295,355 bp and ending after 94,294,900 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
37704 | TGC TGC CTA TCC TTG GAG AC | Mutant Forward | A | |||
37705 | TGG CTC ATG AAG CAA ATT CA | Common | A | |||
37706 | TTG CAA CTG CTA GGC TCA G | Wild type Forward | A | |||
37707 | Fluorophore-1 | TGG GAA AGG AAG CCT GC | Quencher-1 | MUT Probe | ||
37708 | Fluorophore-2 | AAG CAT GAG GTA GCC TTA GAC AA | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
37704 | 0.40 uM |
37705 | 0.40 uM |
37706 | 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 |