Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 82 bp
Wild Type = 93 bp
>chr7:29937735-29937827 93bp GTGGCATTCCAGGGTACAAC CACACGAGTGACAAAAACAACC
Wt Sequence (deletions in lower case; bp changes in brackets with wt first and insertions with carrots ^g^ (g insertion): ATTTAGAGTCTGGGTAGGTGTGCTGACTTCACTTAAGGAAGGTTCGGGACAGTAGCATCTTAGGCACTGTGATTACCAGTGTGATGGGCAGGATTGAAGCTTCCCAACATGCTGAATTCACCATGACAACAGGGGAGGCCAAAACCTCCCTAGCACTCTGTGCTCGCTGGGGAAACAGGCAAGGCTGaagtctcccaggtatgcttgtttattgggtgtcatttttctttatagctgaacaaaattctactatatatacataccactggctctgttttctagtaactgagtataaactgactatattgctaatatgacgtttgcttcattttgtttagaaaatgggcagtgctttttgtaatgtcatctttctgaattatatctgaacaaggcttgttatgtttttcctacaccactagaatggataacttcagtttgagtgatgcgtattgttacagggactagtgacgttcaaagacgtggccattgattttacccaagaagaatggaagcaactggatcctactcagcggaacctatataggaatgtgatgctagaaaactacaacaacttaatcacagtaggtaagataatgtaccttttctaaaacagatgcaattgttaatgttgatgagccttgccttgaggtgccttgagatgttgctgtccctgccccagctttcgaaggaaaatctttggtggcattccagggtacaacattctcagaagttagtacacctggctctcccaggcccaactcagaatccagggttgtttttgtcactcgtgtgtatacccaaaccaatgtcatcccctcccccaccccccaggcccgccactcaccaaacctgaagtgattttcaagttggagcaagaggaagagccttgtgtggtggagagagaagtgctgtggagaccctgtccaggttagtgtgtgtgtgtgtgtggggggtgggggggacccagccaggtaaagcgagaggaccagtaagtgactggcctgagatgtttcctaaggtagattcccacagcgtgtagtaacagttatgtgaaactaagatgttctctttaaaaaaaaaaaaaaaattccctgcacttgggaggcagAGGCAGGTGGATTTCTGAGTTTGAGGCCAGCCTGGCCTACAAAGTGAGTTCCAGAACAGCCAGGGCTATGCAGAGAAACCCTGTCTCAGAAAAAAAATTTTTTTTAATTACGTTTTTTTGTGTACACACATGTG
Mutation details
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
49756 | GTG GCA TTC CAG GGT ACA AC | Wild type Forward | A | |||
49757 | CAC ACG AGT GAC AAA AAC AAC C | Wild type Reverse | A | |||
49758 | CCA AAA CCT CCC TAG CAC TCT | Mutant Forward | A | |||
49759 | AGG CTG GCC TCA AAC TCA G | Mutant Reverse | A | |||
49760 | Fluorophore-1 | CAC CTG GCT CTC CCA GGC C | Quencher-1 | WT Probe | ||
49761 | Fluorophore-2 | ACA GGC AAG GCT GAG GCA GG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
49756 | 0.40 uM |
49757 | 0.40 uM |
49758 | 0.40 uM |
49759 | 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 |