Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr1:33878456-33878568 113bp TGCGTTATAGCAGCTGTGTGT CAGACAACAAAAGACACTTTGC
Mut= 114 bp
Wt= 113 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TAGAAAGTCAGATTCCTGTCTTCTATTTGAAAAGGGAACATGAACCTTTTCGTGTGTCCTGTCCTGAGTTCTGGAGGGTGGAGAACAAAGCTGGGACTATGCGTTATAGCAGCTGTGTGTGGAGGCAGACATGCCGAGGTctgataagaggcattggcatacagaacatgaggtagctgttgaaggaactgcaaagtgtcttttgttgtctgttgtgttttatattttagagagacccatattcaggaaatgctttcctgcctggggagagttccagcgatgaggaaactcccttaatggaactgtccaaggaggaactttgtaacaaaatcgaaagcctgaaagaaaaactaagaagcatccggaaggaaaacagccggcttcgacagtctttggtcatgctgcaaggtaaaacctcaaaaggattggcattttagataaaagggacaaaatgtctaaaatatggtacaactgcagatcaacagttaaaaatggaaaagaaaatcagttcccaagagagctcaaaattacacttcagaaattagtggatagctaaatttaaatatggacatatccggttattgcagagataatttagattgtcttgcaatgtcccaaatattgtattcacttagtagtattcaacagattgctgaagtgaacttaagctacttacatgctgggcctcaGAGGGGGAAAATGAGTAAGGTCAAATAAACACGCTGTGGTTTCCAGATACACTCATCCCACCCTTCCTACCAGGACAGTTCTAGAAAGTGGGAACCGTGTGATCTGCCGAGCCACTGGGGTTACTGGCTCCACAGGCTGCACA
This mutation is a 549 bp deletion beginning at Chromosome 1 position 33,877,979 bp and ending after 33,878,527 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
45242 | TGC GTT ATA GCA GCT GTG TGT | Common | A | |||
45243 | CAG ACA ACA AAA GAC ACT TTG C | Wild type Reverse | A | |||
45244 | CTG GTA GGA AGG GTG GGA TG | Mutant Reverse | A | |||
45245 | Fluorophore-1 | AAG AGG CAT TGG CAT ACA GAA C | Quencher-1 | WT Probe | ||
45246 | Fluorophore-2 | ACA CGC TGT GGT TTC CAG AT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
45242 | 0.40 uM |
45243 | 0.40 uM |
45244 | 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 |