Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr4:40164527+40164617 91bp TGAGGATTGGCTTTCAAGAGA GTAAGCGTCCTGGGGATTAAG
Mut= 96 bp
Wt= 91 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
CAGACATTCAAATTCTGAATGTCTGTTCACTGTACATAGTTTTCACCTTATAGACATTTTAAACTTGTCAATCACTTGGCCCTCTTTCCCTGTTAGAAGAATAATCCGATGTCATGTGTCAAGTTTATCTGAGGATTGGCTTTCAAGAGAGTTGTGAAAAGCAGGTGCCctgtggtttgaggttattaatgctttctaccttaatccccaggacgcttaccattttctatcagagttctcctggaggcagccgttcggaactgtgatgagtttctggtgaagaaaaatgacatcgagaatatcctgaattggaatgtcatgcaacataagaacatagaagtgccgtttaagccagcccgagtcatactgcaagactttacgtgagcctgacacctcacctcacctcacctctctctctctctctctctctctctctctctctctctctcgctcgctcgctcgctcgctcgctcgctcttccctgcccccccctgcctcccttgtcagcctcccaagaaagtttttactgtagcaaaaatgctttgtctctgagaaaccaggagataaaaggaaggcaaggggctcctggaacaagagaagcctgctctgtctctagtagtcaccccgcctcaccagtgacAGGGGTACCATCCTCAGGACTGATCACACTGGGAAGGGTAGAAGACAGCACGCAGGAGGGCTGTGTGGAGTCTCTTGTGCAGGCTGCTGTGCCTGCATACAGTTGTGCCTACCGACCTCAGGGATAGACGAGGTGCTTCGTGAGGCCTGGCTTGCACTCGGCTTGCA
This mutation is a 471 bp deletion beginning at Chromosome 4 position 40,164,567 bp and ending after 40,165,037 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44894 | TGA GGA TTG GCT TTC AAG AGA | Common | A | |||
44895 | GTA AGC GTC CTG GGG ATT AAG | Wild type Reverse | A | |||
44896 | CCT GCG TGC TGT CTT CTA CC | Mutant Reverse | A | |||
44897 | Fluorophore-1 | CTG TGG TTT GAG GTT ATT AAT GCT TT | Quencher-1 | WT Probe | ||
44898 | Fluorophore-2 | AGG TGC CAG GGG TAC CAT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
44894 | 0.40 uM |
44895 | 0.40 uM |
44896 | 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 |