Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr4:101095967+101096071 105bp AGCCTTGGAGGTGGGATTC TGCACCTCGCTTTGTACTTG
Mutant= 108 bp
Wild Type = 105 bp
Wt Sequence (deletions in lower case):
GGACATTGATGGTGATTGTCTCATGGGAGCACAGTCTGTGGGAAGTTGTTAGGAAAAGCTTCCCGGAAGAGCCTTGGAGGTGGGATTCCCGTGTGACCTCaggtggcctgggcagctttggtgactagtttaccccatgctagaagtgtgagctcaagtacaaagcgaggtgcaggctcatgtgggtagtaagtgctctgtttgagtggaacatggggtatgtttaggggtgtgtgtgtgtgtgtgtgtgtgtgtgtgtacacggtgggggaggatagcagtcaagctcggtcatccactaaccactaaccacattgatatacttactgagttttcctggccttgtgtctttcaggaggttcatgagttattacaggactatgagctaaagtattgctatgtggacaggaataaacgaacaggtgagagctcccttccaagcccttgacctcttgaccccgctttcatttttgcatttattgtagagcaggaaagctcgcgctgtcttctccttctggtgctggggattgaactcacggccttcacaggtgctgacaagcatttcaacactgagctctggtctgggtccgcggTGGGGTTCCTAAGTGTTTACATCCCATCAGTGTGGCTCAAGGGTTACTTTCTGAATGTCCTGAACTTTAACCGAGTGTAGCTGGAGCTCTGTGATTCTTAACTATGAATGTTGAATGTTATGTGTTAACAAGTGTTCTCCAGT
This is a 493 bp deletion beginning at Chromosome 4 position 101,095,998 bp and ending after 101,096,490 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
46728 | AGC CTT GGA GGT GGG ATT C | Common | A | |||
46729 | TGC ACC TCG CTT TGT ACT TG | Wild type Reverse | A | |||
46730 | CAC TCG GTT AAA GTT CAG GAC A | Mutant Reverse | A | |||
48132 | Fluorophore-1 | CTG GGC AGC TTT GGT G | Quencher-1 | WT Probe | ||
48133 | Fluorophore-2 | TGT GAC CTC TGG GGT TCC TAA G | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
46728 | 0.40 uM |
46729 | 0.40 uM |
46730 | 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 |