Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr4:116750971+116751094 124bp TCATATTTCTGCATCAGCATCC AAATAAAGCGAAAGCAGCAG
Mut= 112 bp
Wt= 124 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
CAGTGCTCTTGCATTTTGAGCCATCTCAGCCCCCCCCCCCTTTTTTTTTTTGAGACTAGATCTCACAGTGTAGCCCAGGCTGGCCATGTACTCATATTTCTGCATCAGCATCCAGAATGCAAGATTTATTTTATCTAAGCTGTTACTAGACATCCTGGTatgttctgtgtgtggagaccttgctgtcacctacagctgctgctttcgctttatttaactttgtaggtgcgacacagagcttctggtcaggtgatggctctcaagatgaacacgctgagcagtaaccgagcaaacctgctgaaagagatgcagctcatgaatagactctcccaccccaacatcctcaggtaatgggttttccttccttccttccttccttccttccttccttccttccttccttccttccatccaggatcagtaagaggcatcactggatgcctgctgcagtagtactttatctcatggagtccatcttGCTGGGGTTGAGCTAGGCTCTAGGTCTTTTTCTCCCCATTACAACTTAATTTCCTGTAATTAACAATATTTAGTAAGGCAACTTCTCTCAAGTTGCTTTATTCTCATCAGAACATCAGTACTAGCCCATTAGAATAATTGTTTCCTACTGT
This mutation is a 319 bp deletion beginning at Chromosome 4 position 116,751,039 bp and ending after 116,751,357 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
41023 | TCA TAT TTC TGC ATC AGC ATC C | Common | A | |||
41024 | AAA TAA AGC GAA AGC AGC AG | Wild type Reverse | A | |||
41025 | TTG TAA TGG GGA GAA AAA GAC C | Mutant Reverse | A | |||
41026 | Fluorophore-1 | TGT GGA GAC CTT GCT GTC AC | Quencher-1 | WT Probe | ||
41027 | Fluorophore-2 | CAT CCT GGT GCT GGG GT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
41023 | 0.40 uM |
41024 | 0.40 uM |
41025 | 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 |