Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr1:151410810-151410928 119bp TGCAAAGTCACCAGATGTCA TTGGTGCTCACAATGGTCCT
Mut = 119 bp
Wt = 119 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
GCACTGAGCCTCTAGCCCTGTTTCTTATATTTCTCAGTAGTTTAGAATGAGCCTAgttaactctaatgtaatgatatactgcaacagaatgtataacaaatataactaacctaagtttctgtgctgctttatagattgaatgtagaagttttcattcctaaaaagagactaaaattcagttcttcgacccaagcggtatcatatttaaataataatcaaatgacttcacacagttgttcatcaaatggaactaaagatactaaagttaaagattgtaaattgacaaatattggaagtaagcttaattatgaaattaaaaaccatagccgcattaaaattaccaaagatatgaaatctaaacctgtagaccagacaaaggaaaagaattggccttctttgcttattcagaagaagatgaaagaactcaagaaagaaaaaaataataaagacagttctgaagaattggagaaatgcaagaaaaatcaccttcctcagaattataattttagcaatatgatcaaggaatcatttgaatcaggaagaaagaagattagcttcaaaattccaaaaaaatcctctactaccctccagaagcttgtcgaagaaaagatctttactatagattctagcaagtcaaagagtaagcaggaggagaagcaacacttgcaaagtcaccagatgtcattaaatctggccaggcacaaaactgaaaactcattttcagattctacacataagcagtctgtttgtgagaggataagaaaggaccattgtgagcaccaaggaacgagtagttccgattctaaggaaaaccatgcccaggtaatgtataaaatgaagggtttggtttttagtaaaagtatttaaaatactcaggttttagttgcttctgttatgatgataatcacagaggactgattctaacttagtcatgtagataattgtgtttgtgtcctatattattttttgtcattctagcttccttaggataatactgataccacctgGTTTTGGAATCATAGTGCAATATCTGGGTTCTTGTGCTCTGGGACATAGAACTGGACAGAGACCTG
This mutation is a 952 bp deletion beginning at Chromosome 1 position 151,410,586 bp and ending after 151,411,537 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
53252 | TGC AAA GTC ACC AGA TGT CA | Wild type Forward | A | |||
53253 | TTG GTG CTC ACA ATG GTC CT | Wild type Reverse | A | |||
53254 | TGA GCC TCT AGC CCT GTT TC | Mutant Forward | A | |||
53255 | TGC AGG TCT CTG TCC AGT TCT | Mutant Reverse | A | |||
53256 | Fluorophore-1 | CTG GCC AGG CAC AAA ACT G | Quencher-1 | WT Probe | ||
53257 | Fluorophore-2 | CTG GGT TCT TGT GCT CTG GG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
53252 | 0.40 uM |
53253 | 0.40 uM |
53254 | 0.40 uM |
53255 | 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 |