Efficient high-yield isolation of high-quality plasmid DNA for

Efficient, high-yield isolation of high-quality plasmid DNA for mammalian cell transfection Katerina Kourentzi, Craig Branch and Karl H. Hecker Invitrogen Corporation • 1600 Faraday Avenue • Carlsbad, California 92008 • USA Pure. Link™ HQ spin column: a HIGH CAPACITY “mini” column Abstract The Pure. Link™ HQ Mini Plasmid DNA Purification Kit is specifically designed for quick and efficient isolation of transfection -quality plasmid DNA from a wide range of bacterial cell numbers with yields of up to 60 mg of plasmid DNA. Plasmid DNA isolated is of consistently high quality as evidenced by its reliable and reproducible performance in all relevant downstream applications such as restriction enzyme digestion, automated fluorescent sequencing, Gateway® cloning, transformation of bacterial cells, and most crucially in mammalian cell transfection. Introduction Traditionally, isolation of plasmid DNA from bacterial cells relies on alkaline lysis in which cells (resuspended in buffer containing RNAse A) are mixed with an alkaline solution containing detergent, which also denatures the DNA. Addition of a neutralizing solution, usually buffered potassium acetate, causes proteins and genomic DNA to precipitate out of solution, which allows for easy separation by centrifugation or filtration. Circular plasmid DNA simply renatures and remains in solution. Isopropanol precipitation is commonly used to recover the plasmid DNA from solution. Silicabased systems have gained in popularity providing plasmid DNA of adequate purity and quality for many applications at lower cost and higher speed compared to anion-exchange technologies. Plasmid DNA in solution, after the alkaline lysis, can bind to silica-based resin or matrix in the presence of high concentrations of chaotropic salts and be eluted in low salt buffer or water. Pure. Link™ HQ Mini Plasmid DNA Purification Kit, a silica-based spin column kit, was developed for isolation of high quality plasmid DNA (high or low copy number) compatible with restriction enzyme digestion, PCR, sequencing, bacterial cell transformation, and mammalian cell transfection. The kit is based on an alkaline/SDS cell lysis procedure and selective plasmid DNA binding to the glass microfiber (GF) filters. Plasmid DNA bound to the silica-based matrix is subjected to procedures aimed at removing contaminants such as genomic DNA and RNA and eluted with a low salt buffer in a final step. Plasmid DNA (pc. DNA 3. 1/cat) was purified from 1. 5, 3, 6 & 9 x 109 E. coli cells. The Pure. Link™ HQ protocol was performed for each 1. 5 x 109 - cell pellet and batches of clarified cell lysate were loaded onto the column to achieve the noted cell numbers. One elution was performed with 100 ml Elution Buffer. Typical spin columns for “mini” plasmid DNA isolation kits perform similarly with a starting amount of 1. 5 x 109 E. coli cells. When using increased amounts of cells, e. g. 9 x 109 cells, a typical mini spin column capacity is far exceeded. Using the Pure. Link™ column, it was possible to isolate 60 mg plasmid DNA achieving a 35% increase in plasmid DNA yields (Figure 1) over a typical “mini” spin column. Mammalian cell transfection The introduction of foreign genes into mammalian cells by transfection has become an invaluable tool for the expression and study of heterologous proteins. A crucial parameter for successful transfection is the quality of the DNA (supercoiled to nicked forms ratio, RNA contamination, endotoxin content). Plasmid DNA isolated using the Pure. Link™ HQ Kit provides consistently high transfection efficiencies with several mammalian cell lines (Figure 3). It has to be noted that endotoxin levels of the plasmid DNA isolated using Pure. Link™ HQ were significantly lower than the values reported in the literature for plasmid preparations using silica-gel slurries (Qiagen, Product Guide 2003, pp. 119 -120). As expected, the endotoxin levels for the Pure. Link HQ-isolated DNA (< 100 EU/mg DNA) were not as low as for the ion exchangeisolated sample DNA. However, all samples were of transfection quality. As reported in the literature (Fox, et al. , Bio. Techniques, 2000, 29(3), pp. 610 -619), endotoxin levels greater than 2000 EU/ mg DNA are required to significantly inhibit transfection for most of the commonly used cell lines. Plasmid DNA isolation from yeast cells The two-hybrid system is a widely used method for studying protein-protein interactions. A common issue after screening is to confirm “positive”clones in a re-transformation assay. The re-transformation assay is based on plasmid DNA isolation from the positive yeast cells, transformation of the DNA to E. coli cells, selection for the “prey” plasmid DNA, and isolation and analysis of the DNA from several transformants. We have successfully used the Pure. Link HQ Mini Plasmid DNA Purification Kit to isolate high quality plasmid DNA from yeast cells. Digestion with zymolyase, which hydrolyses poly (b-1, 2 - glucose) of the yeast cell wall, was coupled with the Pure. Link HQ standard protocol. The total procedure is simple, fast, and reproducible and can be used routinely to isolate both low-copy (ARS/CEN-based) and multi-copy (two micron-based) plasmid DNA from yeast cells. Isolated plasmid DNA is suitable for PCR amplification and E. coli transformation (Figure 4). Kb Figure 1: Capacity comparison of different plasmid DNA purification “mini” spin columns for increased amounts of bacterial cells (n=3) 1 2 3 4 5 6 7 8 9 3. 0 1. 0 Ma. V 203 Isolation of large plasmids Using Pure. Link™ HQ, the plasmids p 5 L 393 (40 kb) & p 1 Kbplus (19. 5 kb) were isolated from 1 ml overnight E. coli cultures with yields 3 -7 mg/ml of culture and 260/280 ratios of 1. 86 0. 07. The plasmids cut fully with several restriction enzymes under standard conditions (Figure 2). Genomic DNA or RNA contamination was not detectable (< 5%). 1 2 3 4 5 Kb 40. 0 5. 0 1. 0 Figure 2: Restriction analysis of p 5 L 393 and 1 Kbplus isolated with Pure. Link™ HQ. Lane 1: p 5 L 393 / Hind III Lane 2: p 5 L 393 / Ava I Lane 3: p 1 Kbplus / Bam. H I Lane 4: 1 Kb Plus DNA ladder Lane 5: 1 Kb DNA Extension Ladder Figure 3: Transfection efficiency using plasmid DNA isolated with Pure. Link™ HQ or an anion exchange-based mini kit. 0. 3 mg of the plasmid pc. DNA 3. 1/Lac. Z/His were transfected in triplicate into Grip. Tite 293 MSR and BHK 21 cells using Lipofectamine 2000. Expression of b-galactosidase (b-gal) was measured by hydrolysis of o. NPG (48 h post transfection). Transfection efficiency was reported as ng b-gal/cm 2 culture dish area. In this figure, relative transfection efficiencies are expressed as percentages relative to the efficiency obtained with Pure. Link HQ mini kit (=100%) for each cell line (n=3). B 1. 20 Lane 1: 1 Kb Plus DNA ladder Lane 2 -3: p. EXP 32/DP 1 –p. EXP 22 E 2 F 1 Lane 4 -5: p. Hyb. Lex/DP 1 –p. EXP 22 E 2 F 1 Lane 6 -7: p. Hyb. Lex/DP 1 –p. EXP 22 E 2 F 1 Lane 8 -9: p. EXP 32/DP 1–p. EXP 22 E 2 F 1 Figure 4: Restriction analysis of positive clones and bait vectors isolated with Pure. Link HQ DNA after a two-hybrid screen with the DP-1 dimerization domain as a bait (fragment cloned into p. Hyb. Lex/Zeo or p. DEST 32) in Ma. V 203 or B 1. 20 cells. p. DEST 22 was used for cloning the “prey” E 2 F c. DNA library. Isolated DNA from positive cells was transformed to TOP 10 cells. DNA was isolated from the selected transformants and analyzed by restriction with Hind III. Acknowledgements We would like to thank Dr. Sharon Cates for valuable help in planning and performing all transfection experiments, Anna Waters for providing vectors, and Dr. Phillip Gray for the two-hybrid positive clones, all from Invitrogen. Low copy-number plasmid DNA isolation Low copy-number binary vectors are commonly used in Agrobacterium-mediated plant transformation. Prior to plant transformation, A. tumafaciens cells are screened for the presence of the vectors. Minor modifications of the standard Pure. Link™ HQ protocol (increased volume of Neutralization/ Binding buffer, extra wash with 35% Guanidine HCl- 40% isopropanol, two 50 -ml consecutive elutions) allow fast, efficient, and reproducible isolation of the lowcopy vector p. BI 121 (14 kb) from A. tumafaciens that is of suitable quality for restriction analysis (Figure 5) and E. coli transformation. 1 2 3 Kb 12. 0 5. 0 4 5 6 Figure 5: Restriction analysis of p. BI 121 isolated from A. tumafaciens (15 ml culture, OD 600= 1. 0 -1. 5) with Pure. Link™ HQ. Lane 1: 1 Kb Plus DNA ladder Lane 2: p. BI 121 - undigested Lane 3: p. BI 121 /Pst I (original purification protocol) Lane 3 -6: p. BI 121/Pst I (modified purification protocol) Results and Conclusions • Quick and efficient isolation of highquality plasmid DNA (low- and highcopy number vectors) from bacterial and yeast cells • Pure. Link™ HQ column has exceptionally high capacity • Very low g. DNA and RNA contamination • Isolation of up to 40 kb plasmid DNA • Plasmid DNA compatible with all routine downstream applications such as restriction enzyme digestion, transformation of bacterial cells automated fluorescent sequencing, Gateway® cloning, in vitro transcription/translation (data not shown) • Low endotoxin content & high supercoiled to nicked forms ratio provide consistently high transfection efficiencies with most of the commonly used mammalian cell lines. Invitrogen Corporation • 1600 Faraday Avenue • Carlsbad, California 92008 USA • Telephone: 760 603 7200 • FAX: 760 602 6500 • Toll Free Telephone: 800 955 6288 • E-mail: tech_service@invitrogen. com • www. invitrogen. com