Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr7:80897707-80897813 107bp TTCTGTCTACTACATGCAACCAAG GCTGGGGTGGTAAACTCAAC
Mut= 101 bp
Wt= 107 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
CTGCAGCATCTGTCTTTATCAGCATGCCCACTTCTCAGGATACAGACATAGGTGGGTGTACCTGGGGCATCTGAAGAAATTGTTAGCTTAGCAGACATTTTGACTAGTAGAGCCTACAAGTCCGTGCAGTTGCTCCAGGGGCCAAATGCAGACTGGAAAAGCCCTCTGAAGCTTGTCTGAAAGCTGACAGACGCCACAGAAGTTCAGgttttagtcggggttctgtgttcaggcttcctacttgtgacctttctaggtgaccatcatgacctgtggcagcctgggctaagtctgtggctttcttgagttgtgatcctgtccctaaagcaggatgagcctgctcatgtgttggacgagggcagctggtcccagggctcccaagtgggctagagaggaaaggctgctgtagggagggcaagcttccatgtctgctcttaagattccccgtctctctggaacaggttgctggtgaccagtggacttccagggtgttacatacaaatgtggcaagtcagagaggacaacggtgagtaaagttcacggggtggaaggtgaggttttaggaaagtgactcacacactcctgtctcttagcagagccaggaaaaaagcacttaagccaggggacagcacagtttctgtctactacatgcaaccaagatgagttagcatccatagtgtagcagacagGCCTACTAGGGCCTCCTTGAACTTCCTCCAGGAGTTGAGTTTACCACCCCAGCTGCTGGCAGAGCACCTAGAGAGCCAGGACCTCCCTGCTGGACGTGGGCAGAGGACGGGCTGAGCTAGAGCCACTGGGGCACTCAGCT
This mutation is a 482 bp deletion beginning at Chromosome 7 position 80,897,760 bp and ending after 80,898,241 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
46833 | TTC TGT CTA CTA CAT GCA ACC AAG | Wild type Forward | A | |||
46834 | GCT GGG GTG GTA AAC TCA AC | Common | A | |||
46835 | AGC CCT CTG AAG CTT GTC TG | Mutant Forward | A | |||
46836 | Fluorophore-1 | AGT TAG CAT CCA TAG TGT AGC AGA CA | Quencher-1 | WT Probe | ||
46837 | Fluorophore-2 | ACA GAA GTT CAG GCC TAC TAG GG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
46833 | 0.40 uM |
46834 | 0.40 uM |
46835 | 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 |