Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 148 bp
Wild Type =165 bp
Wt Sequence (deletions in lower case) :
ATACACATACAGAGACAGACAGAAACACAGACATACAGAGATACAGTCACAGAGAGCTCAACAGAGACAGAGACAGACAGACAGACACACACACACACAGAGATAGACAAAGACAAATAGAGAGACAGAGATCAAGAGTGCACcagagcctatctggctactgaggagaaaggaatgggactggggaggcccctgtgtttgtgctctccaggcaaggaccacgtgtgcaggttcattggctgtggccgtaacgagaagtttaactatgtggtgatgcagctccaggtgtgtccttggggtccgctcacttgcctctttgcccaagagctggagcgtggtgtgggtggggcatgtggcaggaggcatccgttcttgatgggatgggtacacaggggggcaaggctgggtggtgaatgtggctgagatgggtcttccccctcccaccctgtcccaccctgtcctcccagggccggaacctggctgacctgcgccgcagccagccaaggggcactttcacgttgagtaccacactgcgcctgggcaagcagatcctggagtccattgaagccattcactccgtgggcttcctgcaccgtgacatcaagccggtaagcagtgcgctgagagctcctgtggtctgtgggtcctcgcttctcCACGACAGTGCTGGGCCCTCAGAGCCTCCTCTCTTTGAGCACTTCTTGATCTGTACCAAAGCCACAGCCCCTCTTTCTCGCCTCTTGGCCAAGCCAGGTTGAATGCCCGGCTCTCCATTTGCCTTCATCTCTCACCCATGACCCTGTGTCTTCAGTCTAGAGGTGGCACCTAACTTTCTGT
This is a 513 bp deletion beginning at Chromosome 17 position 46,789,743 bp and ending after 46,790,255 bp (GRCm39/mm39).
chr17:46478769-46478933 165bp GGCAAGCAGATCCTGGAGT CAAGAAGTGCTCAAAGAGAGGAG
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
59172 | GGC AAG CAG ATC CTG GAG T | Wild type Forward | A | |||
59173 | Fluorophore-1 | ACT CCG TGG GCT TCC TGC AC | Quencher-1 | WT Probe | ||
59174 | CAA GAA GTG CTC AAA GAG AGG AG | Common | A | |||
59175 | Fluorophore-2 | AGT GCA CCA CGA CAG TGC TG | Quencher-2 | MUT Probe | ||
59176 | CAG TCA CAG AGA GCT CAA CAG AG | Mutant Forward | A |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
59172 | 0.40 uM |
59174 | 0.40 uM |
59176 | 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 |