Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr19:8873347+8873439 93bp CGTGTGTCACCAGGTCTGGC ATAAGGGAATACGGGCTCCA
Mut= 98 bp
Wt= 93 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TAAAGGCGTGCACTGCTACCACCTAGCTTGTCCTCTTTGGTTTTTCAAGATAGGGTTTCTTTGTATAGTCCTGGCTGTCCTGGAACTAACTCTGTAGACCAGGCTGGCCTCGAACTCAGAAATCTGCCTGCCTCTGCCTTCCAAGTGCTGGGATTAAAGGCGTGTGTCACCAGGTCTGGCCCTGCTTgtcctctttatattactgttttggttttgtttcctttcctctgtcttggagcccgtattcccttattttgttaatttctcagtctaaactcaccttcccactccatgtccaactttacctctttcttctctgcaaaacagaatgttggaacttgtggcacaaaagcagcgggaacgtgaagaaagagaggagcgagaagctttagaacgagaaaagcagcggaggagacaagggcaagagctgtcagttgcacgacagaaactgcaggaagatgagatgcgccgggctgcggaggagcgcaggagggaaaaggctgaagagttagctgccaggtctgaagtctcattggtcgctaattgggaaagaaatgaagatttgccttgtccatgtctgacctattttcttcctgtctctattctagacaaagggttcgagaaaaaattgaaagggacaaagcagagagagccaagaaggtgggtgattagaaagtctgggtatagtggagtatgtgggttaatttccttaaagtgtgcttagcatgatttttttgaggcaattttactctagcccagactagtgtcagactcatagtattctcctgcgtcagccttccaagtactctaactaaaagctgtgaactgccaaacctctgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtttggcccatgtactggagggtgaccctttatacatgctagactagtgtgctccctctgaggtaGTGTTCCATTTTGTCTTTCAGTATGGTGGTAGTGTGGGTTCTCGGTCATCCCCACCAGCAACAGACCCAGGTCCTGTTCCTTCTTCTCCCAGCCAGGAGCCCCCTACTAAGCGGGAGTATGACCAGTGTCGTATACAGGTACTGTTTTCAAGTGACCGTCTTGCTTCTGGAATCCCGTGTTGGCTTATAAAGT
This mutation is a 744 bp deletion beginning at Chromosome 19 position 8,873,374 bp and ending after 8,874,117 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
47742 | CGT GTG TCA CCA GGT CTG GC | Common | A | Common | ||
47743 | ATA AGG GAA TAC GGG CTC CA | Wild type Reverse | A | Wt R | ||
47744 | CCT GGG TCT GTT GCT GGT G | Mutant Reverse | A | Mut R | ||
47745 | Fluorophore-1 | TGG TTT TGT TTC CTT TCC TCT G | Quencher-1 | WT Probe | ||
47746 | Fluorophore-2 | CAG TAT GGT GGT AGT GTG GGT TC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
47742 | 0.40 uM |
47743 | 0.40 uM |
47744 | 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 |