Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr1:36889478-36889588 111bp GCATTTCAGAGTTGCTAAAGTTCTG TGAGGTCAAGACTGGCATCC
Mut= 110 bp
Wt= 111 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
CAGGCGTAGATAGGTATTTTCAAGGCACCTTGCACAGCTGGGTTATTGAAGCTGTCTTAGTATTGATCATGACTACATAGCAAAGCATTTCAGAGTTGCTAAAGTTCTGAAGCATTCGCTGTATTTTCCTCGtaaccattactctcatacaagcagtgtttccttggcttgttctggatgccagtcttgacctcagggccctcacaaatgctaattactctacctaccattggtctacattccccagtaagcaatatattaaaatgtaaaattgtatttactgcaaggcaaacggtgaaattgggtttctgttcctatcagtgatgtttggtttgttttcttgacagcattcattcagtcggagagcataatagaagtcctgcgctttgacgacggagggctactacaggtaattctcttgagtaatatgactattcttctttcctgccttggaaaccagtgtaactggttttcctgaagtatgttttatgaactgtagacagtcagtggagttattgcatgaacctcctactagaattactttttagagtaaacatttttctaaacagTGGACTCCCTGTGTAGTATGGAAGCTGTAGGTAAGTAAGTCAGAGTTTGTGTCCTGTGGCTGTGAGGGTATGTAAAAAAAAAAAAAGCCAGGTCAAATGAGGTTGGGTCTGTGAGCTCTCTGGTTGTTAGTGCTGAG
This mutation is a 437 bp deletion beginning at Chromosome 1 position 36,889,104 bp and ending after 36,889,540 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
43386 | GCA TTT CAG AGT TGC TAA AGT TCT G | Common | A | |||
43387 | TGA GGT CAA GAC TGG CAT CC | Wild type Reverse | A | |||
43388 | ACA GCC ACA GGA CAC AAA CTC | Mutant Reverse | A | |||
43389 | Fluorophore-1 | ACA AGC AGT GTT TCC TTG GC | Quencher-1 | WT Probe | ||
43390 | Fluorophore-2 | CCT CGT GGA CTC CCT GTG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
43386 | 0.40 uM |
43387 | 0.40 uM |
43388 | 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 |