Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 99 bp
Wild Type = 100 bp
>chr10:34297331+34297430 100bp CGACAAAATTACCTGGATTGC TTTTCCTTCTGGGCTGGTG
Mut Sequence (insertions with carrots ^c^ (c insertion):
tttattagacaataggttctattttaaaatttattttgagacatggtttagatactgtcgaaattaattagagacttacgaatagacgtaaaatataattcgacatccatttatgtactcaatttcccagatgagattaaatttacagcatcatttaaaaagacaaaaagaacacacttgatcgacaaaattacctggattgccagttctcgC<c>--3982 bp del--Gttgggcaatgatgaagcatttcacttttattcacagaacaacatgaactgagcagaccaaaaatgattcttttttcctttttagccagcagatgagagactatctgaactggttttgaggctgcacagagagtaactaggggagttgggtgtggtggtacaacct
This mutation is a 3982 bp deletion beginning at Chromosome 10 position 34,297,362 bp and ending after 34,301,343 bp (GRCm38/mm10) with a single base pair insertion (C) at the deletion site
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44468 | CGA CAA AAT TAC CTG GAT TGC | Common | A | |||
44469 | TTT TCC TTC TGG GCT GGT G | Wild type Reverse | A | |||
44470 | TCA TTT TTG GTC TGC TCA GTT C | Mutant Reverse | A | |||
44489 | Fluorophore-1 | CGC TAC GGT TAG TTG CAG CT | Quencher-1 | WT Probe | ||
44490 | Fluorophore-2 | TCG CCG TTG GGC AAT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
44468 | 0.40 uM |
44469 | 0.40 uM |
44470 | 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 |