Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr17:26629464+26629557 94bp GTGTTCATGGAGGGCAAATG CCACTGTGAGGGACAAAGAGA
Mutant= 109 bp
Wild Type = 94 bp
Wt Sequence (deletions in lower case):
GAACTGTTTGGGGTACTGTGACTCATAGCCAAGCAACCCCCTTCTCCCCTACTGGTTGACTTCCCATGTAGCCTGGGTTTCCTGGGCATCGTGTTCATGGAGGGCAAATGGGGCCTCTGTagaagcgggctgctccccactctacatgggtgtgggtaactgctctctttgtccctcacagtggtcgggcttgacatccaagatgagatgggcagacacgaagtgggccacatcgacaattccatgaagatcccgctgaataatggagccggctgccgcttcgagggccagttcagcatcaacaaggtacggagggaggcactgcctgggcccttttcctgccccctgcccactcgaccctaacagagtcagcacatgactgccgagaattttcctgagactgggtaggctctgcattccctgagattctagagcctctgatggtgggttcagaaagagctgcccctcgctggagtggaaattacCAAGGGAGGTGAGACCTGGCTTCAAGTCCTCTTCCTCCTCAGCAAGCAGTGGCCACAGGACATTGGGGTTTACAGCTGCGATGACAGCAATGCCACAGTGGCATCCACCACGGCACAAAGCCCACGGGTGCAAATGAGAGAGCACACTGTTCATTCCAGGCCTGTATACGCACCTTGCATCAGAGTCAGATC
This is a 365 bp deletion beginning at Chromosome 17 position 26,629,494 bp and ending after 26,629,858 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
46707 | GTG TTC ATG GAG GGC AAA TG | Common | A | |||
46708 | CCA CTG TGA GGG ACA AAG AGA | Wild type Reverse | A | |||
46709 | GCA GCT GTA AAC CCC AAT GT | Mutant Reverse | A | |||
46712 | Fluorophore-1 | CTG CTC CCC ACT CTA CAT GG | Quencher-1 | WT Probe | ||
46713 | Fluorophore-2 | CCT CTG TCA AGG GAG GTG AGA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
46707 | 0.40 uM |
46708 | 0.40 uM |
46709 | 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 |