Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr19:40836728+40836843 116bp GGAGGGAGAGTGTATTTGCAG CATATTGTGTAGTGAGAGCTTAAAGAC
Mut= 107 bp
Wt= 116 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GGAGCCTACTCACCAGTCCGCTCCCTTTCAACCAAAACTTTGACCTCCTTGGAGGGAGAGTGTATTTGCAGTGTGGTTTTTCTTAAAGCCATACACCTTCCACTCAActttattgctgcgctcctttcgagcaaagtgtgtctttaagctctcactacacaatatgttaggaaaacaacgttaagtcttgatactctgaagatacgtctatatattaaatattcctcctcttgatctaggagctgaagctgccttcttacaagggccaatctcctcagctgaacctcaggcggtattttgctgatttgattgccattgtgagcaatcgcttcaccctctgcccccctgcccgccatctcgccgtctacctgctggacctgttcatggaccgctatgacatctccattcagcagctacacttagttgcgctttcctgtctgcttctagcaagtgagtatgaactggagtgacccgactcaaggccgacagcgctgacaggatgaaaccggagaactcaccgcattattgtcaaatcccagtcacttcccatttattgcttttaggaaatttatattacaaatttattttatggttgttagttagatattaaaaaatagttcaggcatttttttttaaaacaacaaatgcctaaaccctacctcatcaaGATTGATACAAATACAGATTCTAGGCACCACATACAGTACAGGTTAATTGTCAACTAAACGTGATTTTGAATCTCCTAGGAGACACACTCTGCACATGGGAATTTCCAGAGAGGTTTAACTAAGGAGAAACCTACTTTGAGTGC
This mutation is a 560 bp deletion beginning at Chromosome 19 position 40,836,785 bp and ending after 40,837,344 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
43501 | GGA GGG AGA GTG TAT TTG CAG | Common | A | |||
43502 | CAT ATT GTG TAG TGA GAG CTT AAA GAC | Wild type Reverse | A | |||
43503 | CAA TTA ACC TGT ACT GTA TGT GGT G | Mutant Reverse | A | |||
43504 | Fluorophore-1 | CGC TCC TTT CGA GCA AAG T | Quencher-1 | WT Probe | ||
43505 | Fluorophore-2 | CCT TCC ACT CAA GAT TGA TAC AAA TAC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
43501 | 0.40 uM |
43502 | 0.40 uM |
43503 | 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 |