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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr2:29841378+29841477 100bp TTTGTTCTGTGCCTCCCTCT TCTCCCTGGATGAACAGCTC
Mut = 100 bp
Wt = 100 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
GAACTGAGGTCTTTGACAGATCATCAGCCTCTCCTAATTAGGTTTCTGGGTCCATGTTTCTTTTGCActgggcacagttttggggcctaggaatgagttgggtttgacagtttgttctgtgcctccctctctaggggatatacggaacctccttgtctggatcaagaagaatttgctaaaagagcgaccagagctgttcatccagggagacagtgtgtgagttcccatcccccatccctgagcagggcagagggagggaaggctgtctgagtccacgtcCTGGGGTTCTGATTGTCTCATCCCCCTGAGTTCTCTGGCCCCATAGATTGA
This mutation is a 212 bp deletion beginning at Chromosome 2 position 29,841,335 bp and ending after 29,841,546 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
51535 | TTT GTT CTG TGC CTC CCT CT | Wild type Forward | A | |||
51536 | TCT CCC TGG ATG AAC AGC TC | Wild type Reverse | A | |||
51537 | TGA CAG ATC ATC AGC CTC TCC | Mutant Forward | A | |||
51538 | CTA TGG GGC CAG AGA ACT CA | Mutant Reverse | A | |||
51539 | Fluorophore-1 | ACC TCC TTG TCT GGA TCA AGA AG | Quencher-1 | WT Probe | ||
51540 | Fluorophore-2 | TCC ATG TTT CTT TTG CAC TGG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
51535 | 0.40 uM |
51536 | 0.40 uM |
51537 | 0.40 uM |
51538 | 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 |