Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 83 bp
Wild Type = 85 bp
>chr9:42449749-42449833 85bp TGAGACTCTGGCCATCTCTG TCACAGCAGTAAGCACCAATG
Wt Sequence (deletions in lower case; bp changes in brackets with wt first and insertions with carrots ^g^ (g insertion):GATACAAGTTAGCTGGGACTGAGGTGTAGGCAAGAAGATCGTGACGCGTTGCTGGTGGCTGGCGACAGGCAGCGGGGAGCAGAGCCCAGGAGACTGGAGGGAGGCCAGCACCTCTGAGCTCTTCCTGGCCCAGATTTAACTCTCTTTAGGATTCAAGGTCCCAAGACCCTCTTGGAATGCAGGAGATAGGGTTTGTTTCCTTATGATGCCTGGGGCGAATGTACACAGAC[^g^]aggagcgaggctgagtggagcaagtgttttgtgtttccaataattcagatcgcctcaacctcccgagcgtgctcgtgttgaacagctgcggcattacctgcgcgggagatgaaagagaaatcgcagccttctgtgctcacgtgtcagaactcgatctttctgacaacaagctccaagactggcatgaggtaaagcctatgtaccccacctctgaggggggaaaggcgatgcttgttgcttgcttatgtaacttctgttctgctgactgtggtcctacttgagactctggccatctctgtgttagggtcaacagcaagctgccagctacctctggtgAATTCTCATTGGTGCTTACTGCTGTGACTCTGCTTTTATTGGTGTGCGTAGCGTGGCGACTTTCATTTCAGTCATCAGAAGGTGGATGCGTCTTCAGAAGCTCCGATTGCTCACGTTTCTGTTTCTCAGTCCCCTGTCCTCTGCTTCCACTTCTGTATCTCTGCAGACT
Mutation details
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44567 | TGA GAC TCT GGC CAT CTC TG | Wild type Forward | A | |||
44568 | TCA CAG CAG TAA GCA CCA ATG | Common | A | |||
44569 | GAA TGC AGG AGA TAG GGT TTG | Mutant Forward | A | |||
44570 | Fluorophore-1 | CCT GGG GCG AAT GTA CAC AGA | Quencher-1 | MUT Probe | ||
44571 | Fluorophore-2 | AGG GTC AAC AGC AAG CTG CC | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
44567 | 0.40 uM |
44568 | 0.40 uM |
44569 | 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 |