Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 150 bp
Wild Type = 151 bp
>chr7:3424603+3424753 151bp GCTGCACAGGTCTTTCTGACT AAGCCGAGATTCTGCTGTTC
Wt Sequence (deleted region is in lower case)
CTTGCATTTGGGGCTCAGCTTTTTCTCATTCCCGATCTAGTTGACACTGAGCCTACTACTTCCTTTGAAAGTCTAGGCCCTGCCCCCTGCCCCTTAGGACCCGCTGCACAGGTCTTTCTGACTCCTTGAGGCTCGCCTGAGCTCCAGCGGATCCCATCATCATGatgtggtctaacttcttcatgcaagaggaagaccgacgccggacagctgtgggccggcgtcgtgcccaagaacagcagaatctcggcttgactccggagcgagagggcaagatcaagctggggttgctggtggcaatcgtgggtgctacgctggctgtgctggctgtgggcaccgagttctgggtggaactcaatacatacaagaccaatggcagcgccgtctgtgaggccgcccatttggggctgtggaaggtgtgcatcaagcgactgtggcaggcggatgtgcccgcgggcagggagacctgtggcccAGCTGAGCTGCCAGGAGGTGAGAGGCTGTGTTCTGAGCCCAGATCTGTGTCCCACAGTCAGGGAGAAAGAAACTTTTGTCTGGGGGAAACTTTTGTCTTGTTCTTATCCCCCAAGAATGTTTGTGCCCAAATCAACCTGTGCTCACAGAAACTGGAGAGAAAGGGGGCGGGGAGAGTGAGAG
This mutation is a 311 bp internal deletion beginning at Chromosome 7 negative strand position 3,424,975 bp and ending after 3,424,665 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
37166 | GCT GCA CAG GTC TTT CTG ACT | Common | A | |||
37167 | AAG CCG AGA TTC TGC TGT TC | Wild type Reverse | A | |||
37168 | TTC CCC CAG ACA AAA GTT TC | Mutant Reverse | A | |||
37169 | Fluorophore-1 | TGG TCT AAC TTC TTC ATG CAA GAG | Quencher-1 | WT Probe | ||
37170 | Fluorophore-2 | ATG AGC TGA GCT GCC AGG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
37166 | 0.40 uM |
37167 | 0.40 uM |
37168 | 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 |