Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr7:131326222-131326315 94bp GGAGGCAGATGATGAATGCT GGAATGACAAGCCTCAGCAG
Mut= 86 bp
Wt= 94 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletions in lower case):
GGAGGACAGCTGACAGGGATGTAGGTCAGACAAGCGATGGTTTGAAGTTTGGGAGAGAAGCTTTAAGTATGAGAAAAACTGGCTGTCATGATGTCCCTTAAGAGGGACTTAGATTGTAGGGGCCAGAGCTGAGCCCAGCCAACTGCTGTGCCTTCTCTATTATCctcctgggcctgcaggagcccctggggcacagtcactctctttacttgtgatggaatctgggtttcacatgcatagcaattgtaggagaggaaaagatgggaaaggagcagagagctttaacccagggctggtgtcacatgtatgatgggttccaaccctggcctttctcacaaagccctcataattgtaagaagctcactgagaagggaaattctaagcttgttttgtgtcttttcttaagaatcgcaaaggaggcagatgatgaatgctatattgatccatgcaaggacccccatgagagcatcatcctggccaacagcatccctgctgaggcttgtcattcctaccaggccaacaccattgctgttgcgagttgcggtcccctccagtgttgtaatgtatgtggtgtctatgcagatgttaactccctgccaccctgcctttgtagcataagggaaggactctgacatggcacctatggataaaaggaaatcttcatgatcagtatttcaatttcttactagaatattttcttactcaagccaatttctatagtgtttatgttgtatactatctaacattgtgtttatgtacttttgtataAATgtacaatagtgaaaacaatctctgcctctattatgtttcctagtTATATTTCTTATACCACTATATGTATTACCACCGAGGACCAACCTCAGTCATTTTGGAGTTCTGAAGAATGGGAGGATTGGATTGGCACAAAATCAGAGACTCAGGCACAAATGCAGGTGCAAGCTGACAGGTCACCATTGCTGTT
This mutation is a 648 bp deletion beginning at Chromosome 7 position 131,325,916 bp for 46 bp to 131,325,962 bp retaining 3 bp TTA and then an additional 602 bp deletion ending after 131326566 (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
39592 | GGA GGC AGA TGA TGA ATG CT | Wild type Forward | A | |||
39593 | GGA ATG ACA AGC CTC AGC AG | Wild type Reverse | A | |||
39594 | CCA ACT GCT GTG CCT TCT CT | Mutant Forward | A | |||
39595 | CCA AAA TGA CTG AGG TTG GTC | Mutant Reverse | A | |||
39596 | Fluorophore-1 | TGA TCC ATG CAA GGA CCC | Quencher-1 | WT Probe | ||
39597 | Fluorophore-2 | TCT TAT ACC ACT ATA TGT ATT ACC ACC GAG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
39592 | 0.40 uM |
39593 | 0.40 uM |
39594 | 0.40 uM |
39595 | 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 |