pLenti6.2/V5-DEST载体描述 The pLenti6.2/V5-DEST Gateway Vector is a Gateway-adapted ViraPower II lentiviral expression vector for lentiviral-based expression of a target gene in dividing and non-dividing mammalian cells. The vector has the CMV promoter for driving constitutive expression of the target gene and the PGK promoter for driving long-term, persistent expression of the Blasticidin stable selection marker. pLenti6.2/V5-DEST载体优点 Stable expression
Long-term experiments
High-throughput screening
Accurate titer of functional virus (using Blasticidin method)
Flexible and versatile Gateway recombination cloning technology pLenti6.2/V5-DEST载体特征 Human cytomegalovirus (CMV) immediate early promoter to control high-level expression of the gene of interest
PKG Promoter for expression of Blasticidin selection marker Gateway技术 The Gateway Technology is a universal cloning method that takes advantage of the site-specific recombination properties of bacteriophage lambda (Landy, 1989) to provide a rapid and highly efficient way to move your gene of interest into multiple vector systems. To express your gene of interest using
Gateway Technology, simply:
1. Generate entry clones containing your promoter and gene(s) of interest.
2. Generate an expression clone by performing an LR recombination reaction between the entry clone(s) and pcDNA6.2/V5-pL-DEST).
3. Transfect your expression clone into cells of your choice to transiently or stably express your gene of interest.
To fit all of your expression needs, Invitrogen offers state-of-the-art Gateway destination vectors for expression in E. coli, insect, yeast, or mammalian cells, as well as for production of native protein or N- or C-terminal fusion proteins. All Gateway destination vectors have attR sites for recombination with any attL-flanked fragment, regardless of whether it is an entry clone or an Ultimate ORF Clone. The following table lists a variety of available destination vectors.
Additional materials required, available separately: Gateway entry clone, appropriate Gateway LR Clonase enzyme mix, and reaction buffer. 所需材料 在着手开始实验前你需要准备一下材料： Gateway entry clone, appropriate Gateway LR Clonase enzyme mix, and reaction buffer.
• Purified plasmid DNA of your entry clone(s) (10 fmoles each)
• pcDNA6.2/V5-pL-DEST (20 fmoles)
• LR Clonase II Plus enzyme mix (keep at –20°C until immediately before use)
• 1X TE Buffer, pH 8.0 (10 mM Tris-HCl, pH 8.0, 1 mM EDTA)
• 2 μg/μL Proteinase K solution (supplied with the enzyme mix; thaw and keep on ice until use)
• Appropriate competent E. coli host and growth media for expression
• S.O.C. Medium
• LB agar plates containing 100 μg/mL ampicillin 进行LR重组反应 值得注意的事项： If you use E. coli cells with a transformation efficiency of ≥1 × 108 cfu/μg, a typical LR reaction should give >5,000 colonies if the entire reaction is transformed and plated.
For multiple fragment reactions, typical numbers of colonies (per 10 μL LR reaction) are:
• 2-fragment recombination reaction: 2,000–15,000
• 3-fragment recombination reaction: 1,000–5,000
• 4-fragment recombination reaction: 50–500
Confirming the Expression Clone
The ccdB gene mutates at a very low frequency, resulting in a very low number of false positives. True expression clones will be ampicillin-resistant and chloramphenicol-sensitive. Transformants containing a plasmid with a mutated ccdB gene will be both ampicillin- and chloramphenicol-resistant.
To check your putative expression clone, test for growth on LB plates containing 30 μg/mL chloramphenicol. A true expression clone will not grow in the presence of chloramphenicol.
Sequencing To confirm that your gene of interest is in frame with the C-terminal V5 epitope, you may sequence your expression construct, if desired. We suggest using the following primer sequences. Refer to the diagram on page 6 for the location of the primer binding sites.
