Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 89 bp
Wild Type = 120 bp
>chr2:69199149-69199268 120bp TGTGTGTGTTTGTGTGAGTGC AAGCCCGATGACCTGAGT
Wt Sequence (deletions in lower case; bp changes in brackets with wt first and insertions with carrots ^g^ (g insertion):
CTGGTCCTGACGTTGGCTGACCTTTACCTCAGATTCTCCTGCCCCCAGCTGGAAATGTTGGAATTAAAGTCTGCCTTTATGCTAGCTGAGTTGCTCAATTATTAACCATT[ctgtagcaaagtcaacctgcagacataaactcgaaaatgtggcttatttggtgtcgatagcaactaaaatacagctttgtgctctgttttgcagtgtcttaaaacataatgggtgaagacgaattggcactcttaaatcaaagcatcaatgaatttggggataagttcagaaacag... ...agtatcctgtctcttgtgcctaagatctgcctcaactgtcatttgactcataaggaaaattgtgtttgcacagctctttccatttcagtgtgtcttcttgaggtaacgacctctaaaggctgagagtgtaattttgccacatagatgtatttggggccctaataagtagcatctctcctgggtgacagaacttcctgggagctaaaactct^agctcccaggaagttctgtcacccaggagagatgctacttattagggccccaaatac^]GAGAACACAGTTCTTTGTATTTCCTGTGCAAATGTGATTACCCTAGAGAACTGGAAATGGTACTGTCAGCAGTTCATGGTTACTTTATGTAAAATAACCAATATGTAGTCATTTACTTTCC
Mutation details
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44562 | CCT TTA TGC TAG CTG AGT TGC TC | Mutant Forward | A | |||
44563 | TGT GTG TGT TTG TGT GAG TGC | Wild type Forward | A | |||
44564 | AAG CCC GAT GAC CTG AGT | Wild type Reverse | A | |||
44565 | Fluorophore-1 | CTC CCA GGA AGT TCT GTC ACC C | Quencher-1 | MUT Probe | ||
44566 | Fluorophore-2 | CAC CCT TGT GGA GGT CAA AGG T | Quencher-2 | WT Probe | ||
45430 | TGG GGC CCT AAT AAG TAG CA | Mutant Reverse | A |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
44562 | 0.40 uM |
44563 | 0.40 uM |
44564 | 0.40 uM |
45430 | 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 |