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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr11:95140773-95140865 93bp TTGGCCACCATAGAGTCTCC GAGCTGGGGCCTTTTACA
Mut = 98 bp
Wt = 93 bp
Fam = Mut
Hex = Wt
MUT Sequence (junction in uppercase):
tggcggaacattaagctttccggacctgaatcactttccttctgaagcaaccgcgctggggcccgcaggaaataCCactgagccatctctccagcccaacattagattctcggtatgcctctgctgccagctagctctggggttctggctcaggaacattgtaacagcctgt
This mutation is a 2312 bp deletion beginning at Chromosome 11 position 95140139 bp and ending after 95142450 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
54698 | TTG GCC ACC ATA GAG TCT CC | Wild type Forward | A | |||
54699 | GAG CTG GGG CCT TTT ACA | Wild type Reverse | A | |||
54700 | CAT TAA GCT TTC CGG ACC TG | Mutant Forward | A | |||
54701 | TCT AAT GTT GGG CTG GAG AGA | Mutant Reverse | A | |||
54702 | Fluorophore-1 | ATT ACT GGT GGT GGC CCA G | Quencher-1 | WT Probe | ||
54703 | Fluorophore-2 | CCC GCA GGA AAT ACC ACT G | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
54698 | 0.40 uM |
54699 | 0.40 uM |
54700 | 0.40 uM |
54701 | 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 |