Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr2:26927006-26927115 110bp GGGAGCATCTAAAGCAGGTG AACAGGTCAGAGGAGACAAGC
Mut= 110 bp
Wt= 110 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
AGATAGTATTAGACATTGCCTATGGTCCTGTGCCAGATGTGTGTGACGCTGTCTTTGTCTCTGGGACTAAGTGTTGATCATCATGTGTCTAGACTTACGATGGGAGCATCTAAAGCAGGTGTTGAGTCCTGCAGTCTGCCCTGgcaaggcactggacctgctgagggcctctctgtcttccagagagctggcttgtctcctctgacctgttgtcatgatgaggttgcagccacctcaccagtgtgttctgtggtcttacagttccttcgagtcaccaagcagtacctgcctcatgtggcgcgcctctgcctgatcagcaccttcctggaggatggcatccgcatgtggttccagtggagtgagcagcgtgactatatcgacaccacctggagctgtggctacctgttggcctcatccttcgtgttcctcaacctgctgggacaattgagtaagtggctctctagcctctctagtatacttctggagcatcctgttcccttgactgatactccccattcattgttgttggccattgagaagctatgtgtgaacttgaggtctgggtaccccttcctgggctgaagGCTGATATGACAAACTTAAGAGCTTCTCAGCTGAGATTACAGCTCTGGGTGTTGAAATATGCCAGCCATGGTTTGGTTCTGAGCTTTTGGGTGGATGGTCCATACTCAGTTCCCTTGCACGCATTTCTTTAAACTTGCTCTAGAGATCGGAC
This mutation is a 441 bp deletion beginning at Chromosome 2 position 26,926,633 bp and ending after 26,927,073 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
46105 | GGG AGC ATC TAA AGC AGG TG | Common | A | |||
46106 | AAC AGG TCA GAG GAG ACA AGC | Wild type Reverse | A | |||
46107 | TGG CTG GCA TAT TTC AAC AC | Mutant Reverse | A | |||
46108 | Fluorophore-1 | CAC TGG ACC TGC TGA GGG | Quencher-1 | WT Probe | ||
46109 | Fluorophore-2 | CCC TGG CTG ATA TGA CAA ACT TA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
46105 | 0.40 uM |
46106 | 0.40 uM |
46107 | 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 |