Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr1:172632559-172632661 103bp CGACCAATTTGTGAAGATCG GCCCACTCATTTCCTTCCAG
Mut= 91 bp
Wt= 103 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TACGTGGCCCGCTAGGGCAAGTGCTGAGCGGGGACGGAATCCAGCCCCAGAGGGGGGGTGACCCAATCTCCCGGCAGAGTAGGAAAGGCCAGCCTCGCCCTGAGTAAACTCCCCCCACGATGGgcgtgcaaccccccaacttctcctgggtgcttccgggacggctggccggactggcgttgccccggctgcccgcgcactaccagttcctgctggaccagggtgtgcggcacctggtgtccctgacggagcgcggaccccctcacagtgacagctgtcccggcctcacgctgcaccgaatgcgcatccctgacttttgcccgccgtccccggaacagatcgaccaatttgtgaagatcgtggacgaggccaatgcccggggagagGTCAGCACGCTCGTGAGCCAGAGCGAGAGGGCGGGGCCTGGAAGGAAATGAGTGGGCGGGGCCGGAGCTGGAGTCCGAAAAGAGTGGGAGAGCCTGGGGTTGGGGGCGGGGGTGACAGGGCTAAGTGGGGTGGGAG
This mutation is a 263 bp deletion beginning at Chromosome 1 position 172,632,616 bp and ending after 172,632,878 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
42007 | CGA CCA ATT TGT GAA GAT CG | Wild type Forward | A | |||
42008 | GCC CAC TCA TTT CCT TCC AG | Common | A | |||
42009 | CAG CCT CGC CCT GAG TAA AC | Mutant Forward | A | |||
42010 | Fluorophore-1 | ACG AGG CCA ATG CCC | Quencher-1 | WT Probe | ||
42011 | Fluorophore-2 | CGA TGG GTC AGC ACG C | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
42007 | 0.40 uM |
42008 | 0.40 uM |
42009 | 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 |