Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr1:93811938+93812038 101bp CCTACGAGATGGTGAGTGCAG ACATTTCCATGCACCCTTACA
Mut= 86 bp
Wt= 101 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TGTAGATGAGTTCATAGCTGCCATTAGGCCAGCTTCTCTTTCCCACCTCTCAGCTGGAGAGACCCACCGTGGTACCTGGAAACCTTGTGGGAGTAGCCTCTCTCCCTGAAACCACAGAGAGTCCCACTTCTCAGTCAGAGCAAAGAGTTGCACATGTCTCTGCTGTTgctattcatggaagcatccaggatgtttctatgtgtcatgctgtattgttcactcctttggctatgtgacttaagttgtgcaacaggaaccaggcaaggtgaccatatgggtgtgctgctgtctagaaattgtacagtgacatggccagcgatgctcttgccagcttcctgaatgcttgtcactgcctcttacggaagtcacctgtcctcattagataagaaagcagagatcgacatcctggctgcagagtatatttccacagtgaagactctctcgtcagcccagcgtgtggagcacctccagaagatccagagcgcctacagcaagtgcaaggagtacagtgatgacaaggtgcagctggccatgcagacctacgagatggtgagtgcaggggccagtgcagcctcctgctgccttccgtgcccctgaagGAGAGTGTGCAAGCAAGTGTGTAAGGGTGCATGGAAATGTGTAAGGTGTGTAGAAATGTGTAAGGGTGTGTGAGAGTGTGTAAGGCTGTGTGAGAGTGTGTAAAGGTGCATGGAAATGTGTAAGGTGTGTAGAAATGTGTATCTGGAATAGGAGTTTG
This mutation is a 432 bp deletion beginning at Chromosome 1 position 93,811,567 bp and ending after 93,811,998 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
43741 | CCT ACG AGA TGG TGA GTG CAG | Wild type Forward | A | |||
43742 | ACA TTT CCA TGC ACC CTT ACA | Common | A | |||
43743 | TCC CAC TTC TCA GTC AGA GC | Mutant Forward | A | |||
43744 | Fluorophore-1 | CCT GCT GCC TTC CGT G | Quencher-1 | WT Probe | ||
43745 | Fluorophore-2 | ACA TGT CTC TGC TGT TGA GAG TGT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
43741 | 0.40 uM |
43742 | 0.40 uM |
43743 | 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 |