Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr13:3596711-3596800 90bp GACGGAGTTACTTGGAGCAGA GCCACAGAGTGGGCAATAAC
Mut= 109 bp
Wt= 90 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GAACTCCGGACCTTTGGAAGAACAGTCGGGTGTTCTTACCCACTAAGCCGTCTCACCAGCCCCCTTTCTCCAGCCTTTTAAGGACTTTTTTATAATTATATTTTTGTTTGCATTTGCATTAGAACGTGATAAGCTTAtgtcggtatttattacactgttttatgagtttttgttttttaggccccgggaactagattttaagtatgtaatggacgtgtcttctctgaaagaaagtctaccagaagctgcctttagaagacggagttacttggagcagaaaggtctgttgaaatgtaatggcaccctctGTATACTGGGCTTTCTTCAGTTATTGCCCACTCTGTGGCCCATACATTCATTTACATCTTAAATACTTTTTTTTTCTTTTCTTTTTTTTTTTCAAGACCAGGTTTCTCTATGTAGCCCTGGCTGTCCTGGAACTCACTCTGCAGACCAGGCTGGCCTCGAACTCAGAGA
This mutation is a 171 bp deletion beginning at Chromosome 13 position 3,596,750 bp and ending after 3,596,920 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
41031 | GAC GGA GTT ACT TGG AGC AGA | Wild type Forward | A | |||
41032 | GCC ACA GAG TGG GCA ATA AC | Common | A | |||
41033 | CTC CAG CCT TTT AAG GAC TTT T | Mutant Forward | A | |||
41034 | Fluorophore-1 | AAG GTC TGT TGA AAT GTA ATG GC | Quencher-1 | WT Probe | ||
41035 | Fluorophore-2 | ACG TGA TAA GCT TAG TAT ACT GGG CT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
41031 | 0.40 uM |
41032 | 0.40 uM |
41033 | 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 |