Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 90 bp
Wild Type = 109 bp
>chr4:96769872-96769980 109bp ATGCTTAGACAGACAGCCATGT ATACACAACGCTGGCATGAG
Sequence (deleted region in lower case)
ACTGGCAAAAGAGTTTGACTTTTGTCCATTTCTAGGTGCAAAAGCTTATTTGAGTTATAAATGCGCAATGTTCTCATGGTTAGGGTTCAATTTTGACTGTGTTCACTTCCTGTAGTAAGTGGTCTAGAGCAGATAATCAGATTATTAGGAAGTGAGAATAGAAAAAATAGTCTGAGAAGACAGTGGCATGGTGCTCTCAGGTACATGATGCTTAGACAGACAGCCATGTTTACCACTAAttacatctcacagggctgttcctgctgttcacgcttccatactcacaccttctgcttctcatgccagcgttgtgtatgatgaggagtgttccttatgactgacaaggtgcaaactctcttcacagtaagcagaatgagaagttgaagacagaacctcaaacctcactccagaaacccaggaatgattattcaagacgggtgagcagagacctgccaggacccactgactcttctgagcaacaaataactgctaacccagctgagaaggaagaaagcaagcaccaaagaagcagcctaaaacccgaaagcaatcaaaagttcttaaccagagtattcagtaacaggttcctggatggcagggtgagtaacaagagatggccaagcttttcttggtaactcattagaaaagtgcagaggagaacataatttcatgcatacatatacaaatagaaataagtactttttcatagactagaaattataaatgcttctttttcaacttgaaaataaattttgaggggattttgtgtagtacaattttgtctacaacaaattgagctggtcaacataactgtggcaaACTCACTGATACTCAGCAATGGATACAGTAATGAGCATCACTAGCCAAAGGTCTGAGGTTTATGTTATGTTACGTGATAAACGCCATGATCAAAAGCCACTTAGGGAGGAAGAAGGGGCTTATTTTTCAGACAGCTTATGGTCCATCTTGAAGGTAAATCATAGTATAGAAAGGCAAGAACCTTGAGTCCTGAACTGGAGCAGTGACCACTGAGGGATGGTGCTTATTGGTTTGCTCTCCGTGGCTTGCCCAGCCTGGTCTCTTATATAACTCAGGACCACCTGACCAAGGTAGCACCATTCCAAATGAGC
580 bp deletion beginning at Chromosome 4 negative strand position 96,769,948 bp and ending after 96,769,369 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
37699 | ATG CTT AGA CAG ACA GCC ATG T | Common | A | |||
37700 | CCT CAG ACC TTT GGC TAG TGA | Mutant Reverse | A | |||
37701 | ATA CAC AAC GCT GGC ATG AG | Wild type Reverse | A | |||
37702 | Fluorophore-1 | TCA GCA ATG GAT ACA GTA ATG AGC | Quencher-1 | MUT Probe | ||
37703 | Fluorophore-2 | CAT CTC ACA GGG CTG TTC CT | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
37699 | 0.40 uM |
37700 | 0.40 uM |
37701 | 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 |