Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr17:65717527-65717620 94bp TGAGGCTCCTAAGTCTGTGG ATTTCCACAAGGCACGGTTT
Mutant= 74 bp
Wild Type = 94 bp
Wt Sequence (deletions in lower case; bp changes in brackets with wt first and insertions with carrots ^g^ (g insertion):
CTCTCTCTCATTACTTCTTGTTTAGGGACAAGGGTTCTTAGGGTCATTTTGAGGCATAATAACAGGAACTGAGGCTCCTAAGTCTGTGGAGTTTCGATGATGCAgaatggttgactctcttttcagggcatctttcatgaccaaaaccgtgccttgtggaaatgaacttcacaaattcctcatctgggacaccgctggccaggagcgggtaagtacatgctacttctggctttctagggacaaaat^taaag^GGAGCCAGCTTCTGCATTAATATACACACATGCATGTACACACACATATACATACACACACGTGCACGCACACACATATACATACACACAAGTGCACAC
This is a 142 bp deletion beginning at Chromosome 17 position 65,717,444 bp and ending after 65,717,585 bp (GRCm38/mm10). There is a 5 bp insertion (TAAAG) at the deletion site.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
46722 | TGC ATG TGT GTA TAT TAA TGC AGA A | Mutant Reverse | A | |||
46723 | TGA GGC TCC TAA GTC TGT GG | Common | A | |||
46724 | ATT TCC ACA AGG CAC GGT TT | Wild type Reverse | A | |||
46725 | Fluorophore-1 | TTC GAT GAT GCA TAA AGG GAG | Quencher-1 | MUT Probe | ||
46726 | Fluorophore-2 | TGG TTG ACT CTC TTT TCA GGG | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
46722 | 0.40 uM |
46723 | 0.40 uM |
46724 | 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 |