Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr18:75611651-75611765 115bp CAACTCCCTGGACATGCTG ATGGCAGGAGATCCCACAC
Mut= 107 bp
Wt= 115 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
CACCGTCTCAGTGAGGGCCTGCACTTGCCACAGTGTCCAGGCCTGCAGTCTCCACAGTGTCCTCCTGATGCTCCTGCTACAATATTCCTTTAGGTGGCTCCCTCCAGCCCCTAGCCATGCAGGCTAGCCAGCCTTAGGTGTCAGCGAGGGCCCcagtggccctctcaggcttggacctactgagcctctacctagttgtttccagctggccccataagtaatgccagcccctctctttgtctctttcagaatggcaacaaggacaactccctggacatgctgggcacagatatctgggccgccaatacctttgattccttcaggtaacttcctcctTTTTTGTCAGATGTCCAGGGCTGGTGTGGGATCTCCTGCCATGCATCAGGACTTATGGGGTGGATCTGGTATGGAAACAGATGATGAGGCTGGAGGGGAAGGAGAGACTTTAGCAGAGGCTCCAAGAAAGGAAGAGCCAAAGGGCAGAGGTCAAAGGGAAGATAG
This mutation is a 183 bp deletion beginning at Chromosome 18 position 75,611,693 bp and ending after 75,611,875 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
43374 | CAA CTC CCT GGA CAT GCT G | Wild type Forward | A | |||
43375 | ATG GCA GGA GAT CCC ACA C | Common | A | |||
43376 | TTT AGG TGG CTC CCT CCA G | Mutant Reverse | A | |||
43377 | Fluorophore-1 | AGA TAT CTG GGC CGC CA | Quencher-1 | WT Probe | ||
43378 | Fluorophore-2 | CAG CGA GGG CCC TTT T | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
43374 | 0.40 uM |
43375 | 0.40 uM |
43376 | 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 |