载体特征 pcDNA6.2/V5-pL-DEST载体含有以下元件： • Two recombination sites, attR1 and attR2 for recombinational cloning of up to 4 entry clones when using MultiSite Gateway Pro kits
• The ccdB gene located between the two attR sites for negative selection
• Chloramphenicol resistance gene located between the two attR sites for counterscreening
• The V5 epitope tag for easy detection using Anti-V5 antibodies
• The Herpes Simplex Virus thymidine kinase polyadenylation signal for proper termination and processing of the recombinant transcript
• f1 intergenic region for production of single-strand DNA in F plasmid-containing E. coli
• SV40 early promoter and origin for expression of the Blasticidin resistance gene and stable propagation of the plasmid in mammalian hosts expressing the
SV40 large T antigen
• Blasticidin resistance gene for efficient selection of stable cell lines
• The pUC origin for high copy replication and maintenance of the plasmid in E. coli
• The ampicillin (bla) resistance gene for selection in E. coli 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
