GeneArt™ Site-Directed Mutagenesis System

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- Use 20–25 ng of the target plasmid per 50 µL of mutagenesis reaction as a starting point.	- Create a fresh dilution of 25X SAM from the kit-supplied 200X SAM in sterile, distilled water each time you perform the mutagenesis procedure - 25X SAM is not stable, and loses activity within a few hours after preparation

Upstream tips
- Use 20–25 ng of the target plasmid per 50 µL of mutagenesis reaction as a starting point.
Protocol tips
- Create a fresh dilution of 25X SAM from the kit-supplied 200X SAM in sterile, distilled water each time you perform the mutagenesis procedure - 25X SAM is not stable, and loses activity within a few hours after preparation

Publication protocol

Plasmids and luciferase assay
The genomic sequence spanning 7p14.3 variant was generated from PC-3 genomic DNA using primer pairs as detailed in Supplementary Data . For the identification of enhancer activity the fragment was cloned in pGL4.26 (Promega) in which firefly luciferase is driven by a minimal promoter. The PCR fragment was cloned upstream of the firefly luciferase gene using Kpn1 and XhoI restriction enzymes. Constructs harboring the alternative allele for the study variant (adenine) was created with the GeneArt Site-Directed Mutagenesis System (Invitrogen, Life Technologies) according to the manufacturer’s instructions. The correct insertion of the genomic sequence was confirmed by restriction enzyme digestion and sequence analysis (Eurofins genomics). All plasmids were purified from DH5α bacterial cells using the PureYield Plasmid Midiprep system protocol (Promega). The day before transfection, PC-3 cells (8 × 10 cells) were seeded in 24-well plates. Cells were transfected using TransIT-LT1 reagent (Mirus, TemaRicerca) with pGL4.26-derived vector (350 ng). pRL-SV40 vector (50 ng) (Promega) was used to normalize the transfections efficiency. Construct harboring cytosine allele showed efficiency levels consistent with reference allele. In a parallel experiment, PC-3 cells were cotransfected with pGL4.26-derived vector and pCMV-AR24Q expression vector and/or pCMV6_CEBPB (100 ng, to over-express AR or CEBPB) and treated with 100 nM DHT for at least 16 h. CEBPB or AR silencing was performed by transfection of PC-3 or LNCaP cells with siRNA against CEBPB or AR (20 nM) (FlexiTubeGeneSolution for CEBPB or AR, Qiagen) and Hiperfect transfection reagent (Qiagen) or Lipofectamine 2000 (Thermo Fisher Scientific), respectively. AllStars Hs Cell Death siRNA and AllStars Negative Control siRNA (Qiagen) were used as positive and negative control (Supplementary Fig. ). Forty-eight hours after over-expression or 72 h after silencing, cells were lysed using Passive Lysis Buffer 1X (Promega) and Firefly and Renilla luciferase activities were measured with Dual-Luciferase Reporter Assay (Promega) using the Infinite M200 multi-plate reader (Tecan).



Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing Site Directed Mutagenesis (SDM) Human - Point mutation PC-3 Speckle-Type POZ protein (SPOP) using GeneArt™ Site-Directed Mutagenesis System from Thermo Fisher Scientific.

Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for GeneArt™ Site-Directed Mutagenesis System below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing Site Directed Mutagenesis (SDM) Human - Point mutation PC-3 Speckle-Type POZ protein (SPOP) using GeneArt™ Site-Directed Mutagenesis System from Thermo Fisher Scientific. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.