Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr15:8082689-8082798 110bp CCTTTACTGTTGAGCCACCTG AGAGCTGGGTCTCATCCTGA
Mutant= 102 bp
Wild Type = 110 bp
TGGAGCTGGTCTTAAAGGCTGTTGTGTGTTGCCTGCTTGCCTTTTGGGAACCAAACATGGGTCCTTTGTAAGAGGTCTCACTATGTGGCCCTCTGTCACCCAGGCTTGCCTCCACCATACAGAGATCTACCTGCCTCTTTCTCCCTAGCTTTGGGCTTAACCGTGTGTGTGCCACCACACCATCCTTTACTGTTGAGCCACCTGTGGCTCTGCCTCTGCTTCCCGggggattccattccagatgtgtgccacctctcctgattcttgctctgctcaggatgagacccagctctgtttgttttgggcaggccttctagtcacactgtctttgtaacttaattttcagcctaatattgataaatccgactgatggaaattgtgtttacttttaacttacagaagcaagagaaaaagaggaagaaatgaacagagaaaaagaattaagaaaacaaattgaagatatggagccagcaccgtcaagctcgagtgcagccagagagcgctcacagtcatcatgcaggtgcctgggcttccgcaaggcttctcaacgcccattctccatgtggccataatgcagtgtagtgagaaaagatgaactgtttctccaccgtagacagttgtgtacctaggacatggaaatgctgagtcagctttggagctggggatcttgtgatacagGGTTTTAAGAGAAAGTGATGTAGAAACACTTGTCCCATGCATTTTGTTGAAGTAGAAGCCATTCATTGTAAAGTTTGTTTTTGGCTCCTCTTGATGGCTACGTTGTATAGGCAGGCAGGCAGGCGTCCAACACTGTTCTAGGGATGTGCCAGGTTTGTGATTTTGATCATTTTCTTTCTTGTTTTAAATTTCTTTTTAATTTTTTATGTTTATTTATTTACATTTCAAATGTGATCCCCC
A 453 bp deletion beginning at Chromosome 15 position 8,082,304 bp and ending after 8,082,756 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
45247 | GGC TTC TAC TTC AAC AAA ATG C | Mutant Reverse | A | |||
45248 | CCT TTA CTG TTG AGC CAC CTG | Common | A | |||
45249 | AGA GCT GGG TCT CAT CCT GA | Wild type Reverse | A | |||
45250 | Fluorophore-1 | CCA GAT GTG TGC CAC CTC T | Quencher-1 | WT Probe | ||
45251 | Fluorophore-2 | CTT CCC GGG TTT TAA GAG AAA G | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
45247 | 0.40 uM |
45248 | 0.40 uM |
45249 | 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 |