Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr19:26646876+26646984 109bp AGTTGGGTATCAGAAGTTGACTG GGTGGAGACCTGCATTATGA
Mut = 95 bp
Wt = 109 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
TACATGCACTATGGTATGTACATTTCCCCCCTCTAATAAATAAATAATTGCAGTAATTTTTTTTCTTTTAAAAGAAAAGAAAACTTAGTTGGGTATCAGAAGTTGACTGTCAGTATATGGAatataatttactatgaacccaaacctaaaacattgctgatcatagaggaagaaatcataatgcaggtctccacccaccccacccctgtcttctttcttttttctttctagacttcaagctcgcatcgctcataggatacaagaactggaaagtctgcctggttccttgccaccagatttacgcaccaaagcaaccgtggaactgaaagcacttcgcttactcaacttccaacgtcaggtaatacctttttcccccgtgatgtaggaaATCAATGTGATTGTTGCTAAGGTGTGACCTGGGAGCTTCCTCAAGTCTACCTGATCTTGTTATAAAGCTTAGTGAGATAGTGCGTCTTCAGACTGACAGCTCACGGCTGAGAGTGAGGAGTATGAACCGCTCGTT
This mutation is a 267 bp deletion beginning at Chromosome 19 position 26,646,911 bp and ending after 26,647,177 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
49710 | ACA AGA TCA GGT AGA CTT GAG GAA G | Mutant Reverse | A | |||
49712 | AGT TGG GTA TCA GAA GTT GAC TG | Common | A | |||
49714 | Fluorophore-1 | ATG GAA TCA ATG TGA TTG TTG CTA AG | Quencher-1 | MUT Probe | ||
50858 | GGT GGA GAC CTG CAT TAT GA | Wild type Reverse | A | |||
50859 | Fluorophore-2 | CCA AAC CTA AAA CAT TGC TGA TCA TAG A | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
49710 | 0.40 uM |
49712 | 0.40 uM |
50858 | 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 |