Tips and Additional Information for working with MiniVectors™
Transfection Tips
In our experience, MiniVectors will work with any plasmid transfection protocol. Cell types that are refractory to transfection with plasmid DNA are readily transfected with MiniVectors, thus expanding the opportunities for non-viral DNA delivery. Optimization of transfection protocols is recommended to gain the full benefits of our superior system.
We have had good results with a number of transfection reagents including, but not limited to, Lipofectamine™2000 (Life Technologies), FuGENE™(Promega), Xfect™(Clontech), Effectene™(Qiagen), and GenJet™(SignaGen). We recommend using the standard protocol for the reagent as a starting point, then optimize by varying the ratio of DNA to reagent and the amount of DNA transfected. Because of its excellent transfection efficiency and small size (and consequently more copies per unit mass) you should require considerably less MiniVector and reagent.
We stock a number of different MiniVectors that are ideal for optimization trials. Contact us for more details.
Analysis of MiniVector Size and Topology
You may wish to analyze the MiniVectors by gel electrophoresis to verify the size, supercoiling and integrity. Depending on the cloned insert, the size of MiniVectors can be much smaller than a typical plasmid; therefore, typical techniques for analyzing plasmids may need adjustment. For MiniVectors greater than 1 kb, size can be verified on a standard agarose gel. Smaller MiniVectors will require higher gel percentages (1-2 % agarose) to achieve optimum resolution.
It is important to note that for MiniVectors less than 1 kb, even higher percentage agarose gels lack the resolution to separate supercoiled MiniVector from relaxed MiniVector and linear DNA of the same size. To obtain more detailed information about the DNA structure, we recommend polyacrylamide gel electrophoresis. Individual topoisomers may even be resolved and isolated using this technique. Each MiniVector includes a BbvCI restriction to allow controlled nicking using the nicking endonuclease Nt. BbvCI (New England Biolabs™). For optimum separation of topoisomers we recommend the addition of 10 mM CaCl2 to the running buffer. For more details of electrophoresis with MiniVector DNA, please see Fogg et al., J Phys (2006) or contact us at info@TwisterBiotech.com.
Protein-DNA interactions
MiniVector DNA is uniquely suited for studying proteins binding to a supercoiled, and therefore physiologically relevant, DNA substrate, without the complicating factors of a large excess of non-specific DNA sequence. The small size of MiniVectors mean that unlike plasmids, it may be used in quantitative binding assays. We have had good results using MiniVector DNA in electrophoretic mobility shift assays (EMSAs). Contact us for more details.
Product Quality Control
Twister Biotech is committed to providing a high quality product. As part of our standard quality control (QC) we evaluate and document each batch of MiniVectors according to the following specifications:
1. Product will be clear and free of particulates based on visual inspection
2. DNA concentration will be calculated based on UV absorption at 260 nm
3. Product purity will be evaluated using agarose gel electrophoresis to confirm DNA band location at the expected molecular weight and absence of genomic or RNA contaminants
4. Additional evaluation of purity will be performed via a scan of UV absorption from 220 to 350 nm