Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr9:83839230+83839333 104bp ACACCCCAGAGAACTGGTTG CGCTTGACTATACCGACCAATC
Mut = 109 bp
Wt = 104 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
AGATTCACCTGCCTCTGTCCCCCCAGTGCTGGGATTAAAGGTGTGCAACACCATACCTAGCTTGAAGTTTATTCTCGCTTTTGCTATGTATGATGGAATTTCACAGCAAGAAAGCTGACACCCACAAGAAACCctgtaggttttgaaggcacggattggtgagaccaggaaattagcaaggtatttaagagaactgtagctaacttggaaaaagcacttggaaaggtaacttgtctatttctcagttgtgattttttttttctccttattcttataacttagaaactgttaaccaaattatgagggttgggaacaccccagagaactggttgaatttcttgctgaaactagagaaaaacagctcacctctaaatgacgatcttttaaataagctgattggtcggtatagtcaagcgattgaagtacttcctccagataaatacggccagaatgagagctttgctcgaatacaagtgagacttgctgaactaaaagcgtaagtattagtgaaccacacataaccaactatatatgtactatattacagagaaacagggtaaaaacaggtaatttatattttttaaactagtacttttatttttaaggtaaattttctttctgtggacatagatccctcagagttaCAGTTTGGACATTTCCAAAAATGGTACACAACTTTAATCTAGTTTTTTATTTTCACATTAAAAAAAAAAAAGAATAGTCTATCTCTTTAAAAAGAGAAGTTATCAAG
This mutation is a 511 bp deletion beginning at Chromosome 9 position 83,839,051 bp and ending after 83,839,561 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
50348 | ACA CCC CAG AGA ACT GGT TG | Wild type Forward | A | |||
50349 | CGC TTG ACT ATA CCG ACC AAT C | Wild type Reverse | A | |||
50350 | TGA AGT TTA TTC TCG CTT TTG C | Mutant Forward | A | |||
50351 | ATT AAA GTT GTG TAC CAT TTT TGG | Mutant Reverse | A | |||
50352 | Fluorophore-1 | ACA GCT CAC CTC TAA ATG ACG ATC TTT | Quencher-1 | WT Probe | ||
50353 | Fluorophore-2 | TGA CAC CCA CAA GAA ACC CAG TT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
50348 | 0.40 uM |
50349 | 0.40 uM |
50350 | 0.40 uM |
50351 | 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 |