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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr11:50420446-50420573 128bp GCTTTTGAAAGGAAGCCAGT TCCAAAGGACCAAAGAAAGACT
Mutant= 108 bp
Wild Type = 128 bp
GCTTTTGAAAGGAAGCCAGTGGGGCCCAGGAGGGCCTGCAGCCCATTctcatgacttacactcatgtttccccttttagtcaagaagagtttcattggccaaaataagtctttctttggtcctttggagctggtggaaaaactttgtcctgaagcatcagatatagctaccagtgtcaggaatcttccagaactaaagtgagtaagaagtagtaacgctgagtgtttgcttgcctctctcgggtgtttgccacctCTGAAGTCCTTCCTAACTTAACACTCTGTAGCAGGAACCATGACATGGATTCTGACCCCACCTGTGCTGCTCACTCGTCACATGACCTTGAGCGAGACTT
A 208 bp deletion beginning at Chromosome 11 position 50,420,319 bp and ending after 50,420,526 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
45972 | GCT TTT GAA AGG AAG CCA GT | Common | A | |||
45973 | TCC AAA GGA CCA AAG AAA GAC T | Wild type Reverse | A | |||
45974 | GTG GGG TCA GAA TCC ATG TC | Mutant Reverse | A | |||
45977 | Fluorophore-1 | TCA TGA CTT ACA CTC ATG TTT CCC | Quencher-1 | WT Probe | ||
45978 | Fluorophore-2 | CAG CCC ATT CTG AAG TCC TTC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
45972 | 0.40 uM |
45973 | 0.40 uM |
45974 | 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 |