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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr6:119198583+119198689 107bp AATGACGTCTCAAAGCCAAGA CTTCCCTGCCTCTACCCAAC
Mutant= 98 bp
Wild Type = 107 bp
The common forward primer (primer 65228) anneals over the nucleotide sequence containing mouse genomic variation rs261140489
Wt Sequence : aatgacgtctcaaagccaagaagtttagcatcctgatctcattttcaatctgtAgccgcttggttatgaagcgctcaccaactgcctgttgggtagaggcagggaag
Mut Sequence: aatgacgtctcaaagccaagaagtttagcatcctgatctcattttcaatctgtAGaggatggtgagtttcagactagcctggactacatagtgaagta
A 2545 bp deletion beginning at Chromosome 6 position 119,198,637 bp and ending after 119,201,181 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
65228 | AAT GAC GTC TCA AAG CCA AGA | Common | A | |||
65229 | CTT CCC TGC CTC TAC CCA AC | Wild type Reverse | A | |||
65230 | TAC TTC ACT ATG TAG TCC AGG CTA GT | Mutant Reverse | A | |||
65231 | Fluorophore-1 | CAA TCT GTA GAG GAT GGT GAG TTT C | Quencher-1 | MUT Probe | ||
65232 | Fluorophore-2 | TGG TTA TGA AGC GCT CAC C | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
65228 | 0.40 uM |
65229 | 0.40 uM |
65230 | 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 |