Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr7:46178283-46178382 100bp GGCCTTCTGTAAACCAGTACC ATCTGCACCCCTTTATCCTC
Mut= 99 bp
Wt= 100 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
CGTCTCAAAAATAAATAAATAAATAAATAAATAAATAAATAAAGAGCTGCTGCAGAAGGAAGCTGCTAGTTACTGACAGAGGTAAATGATGTGCATGCCCATATGTCTAGCAGAAACGAAACAGAATGGGTAGACAGTCACACAAGAGGTCAGTGTGGTGGTCCCTGCttaggaccggcctgcaggctcaggctttgtgcctggatatccagttcatatgctcatccctggggaaggaggatttgcatggtctccttatcaccatgctgggcctccaggtgggcgtcctgccttctgcgtctctccccacaggatggggcagccagagctccaaggtacacattcaccatagcacctggctccatttccctggccacaacctgcgctggatccttaccttcatgctgctctttgtcctggtgtgtgagatcgctgagggtatcctgtctgatgggtaagtgagctccaggggagtggggaagctgcaggagaccctccctgtgcctgctcatcggccttctgtaaaccagtacctggtgtgaactgcgccaggccttaggcacatatgagacgcAGGGCCTACCATCAGTCTGGAGGATAAAGGGGTGCAGATTTCTGTACCAGTTCCCAGTGAGACCATTGGAGAGCCTTGTCAGAGTGTCCCAGCTGCTGTGGGCAATACCCGGTCTCTCTGAAAGTGACTGAAGTGCGTGAGGGGACAGTAAATCCTGTCACTTGTTCTTACTGAAGGCCTCTCCTTGGTTCCCA
This mutation is a 406 bp deletion beginning at Chromosome 7 position 46,178,322 bp and ending after 46,178,727 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44885 | GGC CTT CTG TAA ACC AGT ACC | Wild type Forward | A | |||
44886 | ATC TGC ACC CCT TTA TCC TC | Common | A | |||
44887 | AGC AGA AAC GAA ACA GAA TGG | Mutant Forward | A | |||
44888 | Fluorophore-1 | AAC TGC GCC AGG CCT TA | Quencher-1 | WT Probe | ||
47061 | Fluorophore-2 | AGG TCA GTG TGG TGG TCC C | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
44885 | 0.40 uM |
44886 | 0.40 uM |
44887 | 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 |