Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 120 bp
Wild Type = 111 bp
>chr13:104549546-104549656 111bp GGCGTGTGTAAAGCACTTGT GACTGATCTCTCATCTCCACCA
Wt Sequence (deleted region in lower case)
CACAGAAGCCACTGTGCCTAATAAGGGAGGTTTACCAGGTGCTCTTCACGTGTGACTCAGACATGGGACAGTATTGAGTAAAATGGCCATGTCATCACCTTCCCCTTCATCCTGCTAGGATATGCAGGATCTTAGCACAGTGTACTTAAGCATAGACGTGGTCCATCTTAGTCCTGATTACCAGCACTGTCCCTGTCTTGTAAGGCGTGTGTAAAGCACTTGTAAACTAGTAGTGCTCTATttagttgtggaaggcataagatagacttcagtttttcttgctttggtttcttggtggagatgagagatcagtcatcactttctagggttacttttggcgattctgtatttagctatttttatttccttatgtgctgttgagatgtcctcagattgataaacatgaatatcatactaacttttgtttcgtgctttataactattgtgtgtgtttgtgatatactaatatttttaatttagaataaatcactccaacaatgcaatagtaaaacctcctgagatgacacctcaggctgcagctaaggagttagaagaagtaatgaagcgagtacgggaggctcagtcatttatctcagcagccattgaaccaggtaaggccacagtgTTGTTGTACAGGACGTAGTCTAAAGACCATAGACTCTTAGAACTGCAAGGAATTGTAGGATTTCACTGCTCAGTTCCCTCATTGTACAGATCAGAAGACTGAAACTCAAGACATAGTAAGTGGCTTACTCAGAGCAACTCGGCACAAACCAGCTTTGCCCTAACACTGGGCCATGCATTTTAGCCACAGTCAGTGAGCGCTCTAGGAAAACAAGTCAGTGCTGAGGCTGCTCTGCTCAACAGCAGGGTGGGGTGTTTCTGCGAGGCTAGAAAGGAAGGA
384 bp deletion beginning at Chromosome 13 negative strand position 103,759,538 bp and ending after 103,759,155 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
38341 | GGC GTG TGT AAA GCA CTT GT | Common | A | |||
38342 | ATG AGG GAA CTG AGC AGT GA | Mutant Reverse | A | |||
38343 | GAC TGA TCT CTC ATC TCC ACC A | Wild type Reverse | A | |||
38344 | Fluorophore-1 | CCA TAG ACT CTT AGA ACT GCA AGG A | Quencher-1 | MUT Probe | ||
38345 | Fluorophore-2 | TGG AAG GCA TAA GAT AGA CTT CAG T | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
38341 | 0.40 uM |
38342 | 0.40 uM |
38343 | 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 |