Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr5:134196569+134196669 101bp TGGAGAACAAAGCAGGGATCT GCTGCCATACTTGGCTCAG
Mut= 106 bp
Wt= 101 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
CAGTGTCAGCCGTCACAAGAGGGATAGAGCCATCATGGCAGGAGAGCCAGCAATGGGCAGAGAAAGGGTGGGGCCAGGAGCGGGAAGCTGGCTGGTCACATTGCAGCCACACGCAGGAAGGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAAAGAGAGAACGCGCAAGCGCGTTAGTTCTTTGGGCTCTTGATGTATGTTCAAGCTACATGCTCCTGTGTCTATTGCCCTAGATGAAGCtggtggtagaaagtagttctgagagtcccagttaccatctcttacctcctgtcctaggtaaagccttggggacaacagccatggtccctgttccctacgagcagatgctacaggatgaggcagctgtggtggttcggggccttccggaaggcctggccttccagcacccagacaattacagccttgccaccctgaagtggatcctggagaacaaagcagggatctccttcgctgtaaagaggtaatctgctccggagCCTGACAGCCTGTTTAGAGAGAAACTGGCCTGAGCCAAGTATGGCAGCACAGGCCTGCAACCTCGGTACCGGGAGACTGAGACAGGAGAGTCACTGAAAGTTTAAGGCAAGCCCAAGTTACAGTGAGTTCAAGGACAGCCTGGGGTATATACCTAAATCCTGTCTTTTTTCTTTCTTTCTTTCTTTTTTTTTTTTTTAAGATTTATTTATTTCATGTATGTGGGTACAACTGTCGCTGTCTTCAGACACACCAGAAGAGGGCATCAGATC
This mutation is a 257 bp deletion beginning at Chromosome 5 position 134,196,365 bp and ending after 134,196,621 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
47737 | TGG AGA ACA AAG CAG GGA TCT | Wild type Forward | A | |||
47738 | GCT GCC ATA CTT GGC TCA G | Common | A | |||
47739 | TGG GCT CTT GAT GTA TGT TCA | Mutant Forward | A | |||
47740 | Fluorophore-1 | AAA GAG GTA ATC TGC TCC GGA | Quencher-1 | WT Probe | ||
47741 | Fluorophore-2 | CCC TAG ATG AAG CCC TGA CA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
47737 | 0.40 uM |
47738 | 0.40 uM |
47739 | 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 |