Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr7:135518418-135518513 96bp AGCATCAGCAACCCTTACCA GTGCACATGAATCTTCTCAGGA
Mutant= 103 bp
Wild Type = 96 bp
Wt Sequence: GACACCATGCCTGCTCTAGCTCAGCTGTATTTGTATTCTGCACTGTGCACTCACCAAATGTTGCTTTAGTTTTAGGGATATTGGACAATTcttcccagaagtctttgcacctggtggtcatcctcctcctgatcctgagtctggcagcttccgtgctgagctcagtgttcaccttctacaacagcatcagcaacccttaccagacgttcctgggacctatgggtgtctatacctggaatggactcagCGGTGAGTTTCCTGAGAAGATTCATGTGCACACACATGCATACACATACATATGCATACATATGTACACCTGCACACATAGATGTATACACA
Deleted Sequence: cttcccagaagtctttgcacctggtggtcatcctcctcctgatcctgagtctggcagcttccgtgctgagctcagtgttcaccttctacaacagcatcagcaacccttaccagacgttcctgggacctatgggtgtctatacctggaatggactcag
157 bp deletion beginning at Chromosome 7 negative strand position 135,518,605 bp and ending after 135,518,449 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
37617 | AGC ATC AGC AAC CCT TAC CA | Wild type Forward | A | |||
37618 | GTG CAC ATG AAT CTT CTC AGG A | Common | A | |||
37619 | GCT CAG CTG TAT TTG TAT TCT GC | Mutant Forward | A | |||
37620 | Fluorophore-1 | CCT ATG GGT GTC TAT ACC TGG AAT | Quencher-1 | WT Probe | ||
37621 | Fluorophore-2 | CTG TGC ACT CAC CAA ATG TTG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
37617 | 0.40 uM |
37618 | 0.40 uM |
37619 | 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 |