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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr11:104693186+104693341 156bp ATTTAAGCCGGGTGATGGT CTCTGTACACGAATAACGATGTTTC
Mut = 150 bp
Wt = 156 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
GGCCATGATTTAAGCCGGGTGATGGTCCGTGgagagcttagcatcctatttggaaatttccacaatcaaagatcgagaggaagataaagccgggaaaacaactcagcagttattctaatttctgtgccatttagatatgaaacatcgttattcgtgtacagagaagaaaaacctacagagttctcaggatcaaaaaaactcaggagaaagaaaagtgtgcaagtacagcttcatagtaagagaacggaggcgagtctgaacctggcaagtgtcccttgtccccagcccccaaagcttctgGATGTTTTAGTTCAAGTGTTGAAATGTTCCTATTT
This mutation is a 269 bp deletion beginning at Chromosome 11 position 104,693,210 bp and ending after 104,693,478 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
51834 | ATT TAA GCC GGG TGA TGG T | Common | A | |||
51835 | CTC TGT ACA CGA ATA ACG ATG TTT C | Wild type Reverse | A | |||
51836 | GGA ACA ATC AGA GGG TGG AC | Mutant Reverse | A | |||
51837 | Fluorophore-1 | CTT AGC ATC CTA TTT GGA AAT TTC CAC | Quencher-1 | WT Probe | ||
51838 | Fluorophore-2 | CGT GGA TGT TTT AGT TCA AGT GTT GA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
51834 | 0.40 uM |
51835 | 0.40 uM |
51836 | 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 |