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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr13:93098350-93098439 90bp TTCACCATAGGTTATCAGATGAGG GAACTGCCACCATGGAAAAG
Mut= 93 bp
Wt= 90 bp
Fam=Mut
Hex=Wt
Wt Sequence :
ttcaccatagGTTATCAGATGAGGATGAGGAGGGCAAGACCAAGCAGGAGTGTATCGTCTCTGACCCTTCCTTTTCCATGGTGGCAGTTC
This mutation is a 9281 bp deletion beginning at Chromosome 13 position 93,089,133 bp and ending after 93,098,413 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
43445 | TTC ACC ATA GGT TAT CAG ATG AGG | Common | A | |||
43446 | GAA CTG CCA CCA TGG AAA AG | Wild type Reverse | A | |||
43447 | TGT GAA AGC AAA CCC AAT ATC | Mutant Reverse | A | |||
43448 | Fluorophore-1 | AAG ACC AAG CAG GAG TGT ATC G | Quencher-1 | WT Probe | ||
43449 | Fluorophore-2 | AGG TAA ACC AAC TTA ACA CAC ACA AAT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
43445 | 0.40 uM |
43446 | 0.40 uM |
43447 | 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 |