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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 129 bp
Wild Type = 137 bp
>chr7:97966176+97966312 137bp AACCCATTCTTGTCTTTACCC CACTCAGCACCAAAATGGAC |
Wt Sequence (deletions in lower case):
AACCCATTCTTGTCTTTACCCTTAATGTAGATGCTATTACTGATTCCTCCTATATTTTAATCATTgaaatattttttccagggttgtttatctggggacaggatgctggttcttttggtccattttggtgctgagtgcagctgggatcatggttgcctacactaccttgcttttggtgagtgatgcttctcatttttttcctccttaaacagcctcatgggctactccttcctgtgctagtgtatctatctgtagtccttgatattagtagagttggaatttacaccatctccttcaatttcatgcacaagGAGTGGCTGAAGGAGATGTATGCTATTTAGAGTATGAGTCTAATAAAGCTGACCCTTGAGCAGG
This mutation is a 246 bp deletion beginning at Chromosome 7 position 97,966,241 bp and ending after 97,966,486 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
65952 | AAC CCA TTC TTG TCT TTA CCC | Common | A | |||
65953 | CAC TCA GCA CCA AAA TGG AC | Wild type Reverse | A | |||
65954 | CCT GCT CAA GGG TCA GCT T | Mutant Reverse | A | |||
65955 | Fluorophore-1 | TTT CCA GGG TTG TTT ATC TGG | Quencher-1 | WT Probe | ||
65956 | Fluorophore-2 | TTA ATC ATT GAG TGG CTG AAG GAG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
65952 | 0.40 uM |
65953 | 0.40 uM |
65954 | 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 |