Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr11:100632619-100632710 92bp AACCCCTCAGAGCAAAGGTC TGGTTGAAGAGCCAGAGTCA
Mut= 91 bp
Wt= 92 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletions in lower case):
TGAGTGAGGTGCTGTGACTTGCCTAGAGATGCAGAGCTTACATAAATGGCCAAGTTTGATTCTGAGGGCAGGCTGCCTGCATGGGAGCCCCTGTGTGAGAACAAGCTCTGTTGTAAACCTCAGGGCTCTATACATGTGGCAATGATGGTGACACTGACATCACCCCTTTGTGGCTGAGCCTTGTGTTCGTCCCTTTGACCACATATTCCTCTGGCCTATCCCCCTACTCAGCCTCCctcctagatgaggcatgagacccacgggaagaagatgacccctcccctccctcttccagaaccaggctgaggctcattacaagggccacagacacgccagaaaactcaaggctgtggaagctgccaagagcaagcagaggcctcgaaacccgaccacaaacgggacagtggtgtcttcagcctcacctccagccagtggaagccctggaacccctcagagcaaaggtcagtcttgatttcaagttcacacaacgaggCTGGCTTCTGACAGCGGCTTGAGTGACTCTGGCTCTTCAACCATCTTTCTTTCCTTTTTCTTTTGTTTGGTTTTTCAAGACAGGGTTTTTCTGTGTAGTCTCAGCTTTCCTGGAACTCACTCTGTAGACCAGGCTGGCCTTGAACTTATAGAGATCCATCCACCTGCCTCTGCCTCATGACAGTGCTGGGATTAAAGGCATGCACCACTACTACCTGGCTTTTT
This mutation is a 257 bp deletion beginning at Chromosome 11 position 100,632,662 bp and ending after 100,632,918 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
64400 | AAC CCC TCA GAG CAA AGG TC | Wild type Forward | A | |||
64401 | TGG TTG AAG AGC CAG AGT CA | Common | A | |||
64402 | GTC CCT TTG ACC ACA TAT TC | Mutant Forward | A | |||
64403 | Fluorophore-1 | CAA GTT CAC ACA ACG AGG CTG | Quencher-1 | WT Probe | ||
64404 | Fluorophore-2 | CTC AGC CTC CCT GGC TTC TG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
64400 | 0.40 uM |
64401 | 0.40 uM |
64402 | 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 |