Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr10:42924204+42924348 145bp CTAGGTGGCAGTGATCCTAGA AGATGTCAGCCTGCCTTTGC
Mut= 135 bp
Wt= 145 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GTCTTCACCAGTTCCATTAGTGTAAACACCATCTTCTCACGGTGACGTTTCAGGAAGACCCTCACAGTGGTGGCTACTCCTTCCGATCCCCATCTGCATTGTCTGATGGTTTGCTACAGGAAGTCACTGCTGAACAGCTAGTCTGAAACCTGGAGAGAGCTCATGCTGTGAGCTCATCAGAGAATGGGAACAGCACCATGGCCTATCTCCTGGTGGATACAGGGAGAGCCTACAGCTGGGATTCTACCTAGCACAGAGCTGTGGGCAGAGCACTGCCCTGCAGTCTGTGTTCTAATCTTGGGAGCTTgtgtgtcttaactgttcatgcctctctgtgttcctacacacgttgcctggtgctgtcatttcccgggcagtgtcaatgtaagggtacagagctctcctctgaggataatccaaatacaaattttgaacatgaacttgaatctgaagctctgttcccaaagctgaggagtaggaaggtgcaggagtgtgaaggtgtagagagagttagcaggcagccaggatggtcagggtggtatagagagcatttttagccatttgctgattgaactgactctcctgcagatcaagtctccagtgctcatgactcccttggccctgtcacctccacggagtaccccggaacccgatctcagctccatccctcaggatgcagccaccattcccagcctggtggtcccagaggctctcacaggtacgtctgccaaaggacttctaggtggcagtgatcctagagtaacccggcgtcctttcatctaatggttactttatgaatctaagagtttctctgaggtacttatttcactggGTGTGATGACACATGCCTTTAAACCCAGGAGGCAAAGGCAGGCTGACATCTGAGTTTAGGGCCAGCCTGGTCTACAGAATGAGTTCCAGGATAGTCAGGGCTACAGAGAGAAACCCTGTTTCTGAAAAGAAAATATAAAGAGAGAAAGGAAGAAAAGAAAAGAAAAGAAAAGAAAAGAGAAGAAAAGGAAAGGAAAGGAAAGGAAAGGAAAGGAAAGGAAAGGAAAGAAAGGAAAAGGAAAGAAAGGAAAAGGAAAAGGGAAAAGAAAAAGAAAAAGAAGAACAAAAATTTCCTGAGATAGCTCAAAGAAAGAT
This mutation is a 525 bp deletion beginning at Chromosome 10 position 42,923,773 bp and ending after 42,924,297 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
48694 | CTA GGT GGC AGT GAT CCT AGA | Wild type Forward | A | |||
48695 | AGA TGT CAG CCT GCC TTT GC | Common | A | |||
48696 | GAG AGC CTA CAG CTG GGA TTC | Mutant Forward | A | |||
48697 | Fluorophore-1 | CCG GCG TCC TTT CAT CTA A | Quencher-1 | WT Probe | ||
48698 | Fluorophore-2 | CTT GGG AGC TTG TGT GAT GAC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
48694 | 0.40 uM |
48695 | 0.40 uM |
48696 | 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 |