Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mut = 98 bp
Wt = 103 bp
Fam = Mut
Hex = Wt
>chr9:28813184+28813286 103bp GCCTGAGAGGCAAGTGTAGT CAAATATCCATTCGCTGTGA
Wt Sequence (deletions in lower case):
CAGCAAGTGGCAACTGTGACGAATTTAGAATGTATGTGACTGCCTGAGAGGCAAGTGTAGTATCTTCACATTTAAATGTAAcatcttcctaaaatgaatggttggttgactaaggggaggggtgtcacagcgaatggatatttgtcttctctggaattaagttttaatttatttattttttctgtctgtcttgcttttatttctccagaccctccctatatatctaaagccaagaacactggtgtttcagtgggtcagaagggcatcttgagctgcgaagcctctgctgtcccgatggctgaattccagtggttcaaagaagataccaggtaccttgaaggaactgggggagctctgacacagagctgattaatcatgcatcccccagaaggagggtcacaaactccaggggtaaaataaacaagataaaaacatgctataacctccttttaatggagtcttcttttagaatcagaagtgagaaagaaaaaaataaatagagggagctgagaactcaacaaaATGGATTGTCCTTGTTTAAGAATCTTGGATTACAAAGTTTGGAGCTGAGACAAAAGGA
This mutation is a 441 bp deletion beginning at Chromosome 9 position 28,813,224 bp and ending after 28,813,664 bp (GRCm39/mm39).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
57951 | GCC TGA GAG GCA AGT GTA GT | Common | A | |||
57952 | CAA ATA TCC ATT CGC TGT GA | Wild type Reverse | A | |||
57953 | TCC TTT TGT CTC AGC TCC AA | Mutant Reverse | A | |||
57954 | Fluorophore-1 | AAT GAA TGG TTG GTT GAC TAA GGG | Quencher-1 | WT Probe | ||
57955 | Fluorophore-2 | AAT GGA TTG TCC TTG TTT AAG AAT CTT G | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
57951 | 0.40 uM |
57952 | 0.40 uM |
57953 | 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 |