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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mut= 94 bp
Wt= 103 bp
Wt Sequence (insertions with carrots ^^ ):
AAGGGCAAGAGTAAGCTAAAGTGAGTGTTCCTGCTGCCCCGTGGGGTGCTGGGAACAGTACACTGCGGGATAAAGTGACTGCTCTCTGCTGCCCC^ataacttcgtatagcatacattatacgaagttat^TGGGGTGCTGGAGATAGCACACTCCTGTGAGGTTATATTCGTGTGAGC
Loxp Site 3' end of Exon 3
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
39130 | TCT GCT GCC CCG ATA ACT T | Mutant Forward | A | |||
39132 | GCT CAC ACG AAT ATA ACC TCA CA | Common | A | |||
40970 | Fluorophore-1 | GCT GCC CCG TGG GGT | Quencher-1 | WT Probe | ||
40971 | Fluorophore-2 | TCG TAT AGC ATA CAT TAT ACG AAG TTA TTG GG | Quencher-2 | MUT Probe | ||
40972 | TGG GGT GCT GGG AAC AGT A | Wild type Forward | A |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
39130 | 0.40 uM |
39132 | 0.40 uM |
40972 | 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 |