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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr11:99978999-99979098 100bp AACGGATGGTTCTTGCAGAC TGGTGGGCACAGTTGGAA
Mutant= 109 bp
Wild Type = 100 bp
The common primer (primer 65204) anneals over the nucleotide sequence containing mouse genomic variation rs241097027.
Wt Sequence aacggatggttcttgcagactcccagcctggctagtaggaacaccATGGCAGACAGCTGTTGCCCTGAAAACCCCACAGCTGTTCCAACTGTGCCCACCA
Mut Seq: aacggatggttcttgcagactcccagcctggctagtaggaacaccaTAtggacccacttcttgacaacctagctgtgagtgcagcctccccaaggccaaacttcatgat
A 1010 bp deletion beginning at Chromosome 11 position 99,978,042 bp and ending after 99,979,051 bp (GRCm38/mm10). This mutation deletes 1010 bp from ENSMUSE00000665201 (exon 1) and is predicted to generate a null allele.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
65204 | AAC GGA TGG TTC TTG CAG AC | Common | A | |||
65205 | TGG TGG GCA CAG TTG GAA | Wild type Reverse | A | |||
65206 | ATC ATG AAG TTT GGC CTT GG | Mutant Reverse | A | |||
65207 | Fluorophore-1 | AGG AAC ACC ATA TGG ACC CAC | Quencher-1 | MUT Probe | ||
65208 | Fluorophore-2 | CAG ACA GCT GTT GCC CTG A | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
65204 | 0.40 uM |
65205 | 0.40 uM |
65206 | 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 |