Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr14:60046746-60046856 111bp TGCCCTCCTACAAAGGACAT CTGTACTTGGCCGTACTGGA
Mut= 111 bp
Wt= 111 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
CCGAATGAACCTGAATACACACATGTTGCTCTTAGCATGTGGCTCCCAGATGGGAGTCACTGGTCTCGTCACCCCATGGGAAAGTGGAATGTCCATATCAAAGATTGAATTGCAAACACATTGGAAGTGTCCCTGGTCAGgtgtgccctcctacaaaggacatgcagttggatgtcactttgctgttatttaagggttacctctttaactaaaagtcgtacttgtctttgtacttccagtacggccaagtacagcgtgttgacatttctacctcgattcctgtatgagcagattaggagagctgctaacgccttcttcctcttcattgccttattacaggtaatggtttttaaggacttaaacattgcaagctcaattatgtgcaaaatgagattctttagtaagtgcctttattggaaaagtgttagaatatttacatagtgtccttcctcaaattatcactaggggggacagacctgatcttggtataaaacaaacgtgggagGGAAGTCTTTGAAAAAAGAAGTTACAAAAGGGGAGCAGAAACTGGTTCTTGTGGGTCCTACCTTGTCATGTTTACCTTTTCTTGTTATTTTTAAAATTATTAATGCACATTCATTATATTAGACATGATATTTCACTGTGACACTTCATACATATATACCATTCATG
This mutation is a 365 bp deletion beginning at Chromosome 14 position 60,046,495 bp and ending after 60,046,859 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
45038 | TGC CCT CCT ACA AAG GAC AT | Wild type Forward | A | |||
45039 | CTG TAC TTG GCC GTA CTG GA | Wild type Reverse | A | |||
45040 | CCA TGG GAA AGT GGA ATG TC | Mutant Forward | A | |||
45041 | CAG TTT CTG CTC CCC TTT TG | Mutant Reverse | A | |||
45042 | Fluorophore-1 | CAG TTG GAT GTC ACT TTG CTG T | Quencher-1 | WT Probe | ||
45043 | Fluorophore-2 | TGT CCC TGG TCA GGG AAG T | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
45038 | 0.40 uM |
45039 | 0.40 uM |
45040 | 0.40 uM |
45041 | 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 |