Figure 1: E. Coli lysate transfer using liquid handling automation Figure 1 - E. coli lysate transfer using liquid handling automation. Following the manufacturer s procedures, a 96-well plate miniprep is carried out by resuspenion, lysis and neutralization steps. Subsequently, attempts at using liquid handling automation to transfer plasmid containing supernatent to magnetic beads or vacuum plate resulted in clogged pipette tips, inconsistent fluid transfer and transfer of precipitants. As shown in this picture, the nature of the sample consisting of plasmid DNA in the presence of cell debris, particulates, precipitated protein and precipitated genomic DNA creates a difficult task for automation.
Figure 2: PhyNexus Lysate Direct PhyTip Columns for plasmid DNA Purification A B Figure 2 - PhyNexus Lysate Direct PhyTip Columns for Plasmid DNA Purification. The solution for obtaining reproducible, walk-away, automated plasmid minipreps utilizes technology from PhyNexus. (A) These novel columns are derived from pipette tips with a thin, frit screen at the end to retain the Plasmid DNA Resin. The design of the column, selection of the frit screen material and the chromatography resin packed in the column all contribute to a reliable solution for capturing plasmid DNA in the presence of cell debris, particulates, and precipitants. (B) The format of the pipette tip column allows for utilizing liquid handing automation found in high throughput laboratories to process up to 96 samples in parallel. The Lysate Direct PhyTip Columns for Plasmid DNA Purification is also compatible with the MEA Personal Purification System available from PhyNexus.
Figure 3: Quick procedure for plasmid DNA purification Resuspend/Lyse/Precipitate E. Coli sample 12x Bind Plasmid DNA 2x Wash Columns (3X) Air Dry Columns on vacuum Standard Tips PhyTip Columns PhyTip Columns PhyTip Columns 1x Elute Plasmid DNA Figure 3 Quick procedure for plasmid DNA purification. E. Coli cells were harvested by centrifugation and bacterial pellets were resuspended, lysed, and precipitated by pipette mixing using standard wide bore tips. Following precipitation, the plasmid DNA was captured on PhyTip columns containing silica based resin. Twelve cycles of back and forth flow was used to bind the plasmid DNA in the presence of precipitants. After binding, PhyTip columns were washed with 3 separate aliquots of wash buffers with 2 cycles of back and forth flow each. The PhyTip columns were air dried on a vacuum block for 3-5 minutes to remove residual ethanol from the wash buffer. Lastly, plasmid DNA was eluted from the PhyTip column with 1 cycle of back and forth flow.
Figure 4: Analysis of plasmid DNA purified with Lysate Direct PhyTip columns Fig 4A Sample Yield Conc A26/A28 A26/A23 1 18.5 21 1.97 2.12 Fig 4B 2 19.4 28 1.98 2.2 3 18.5 24 1.95 1.97 4 16.5 188 1.97 2.12 5 17.7 195 1.95 2. 6 19.1 26 1.94 1.96 7 19.5 26 1.96 1.96 8 19.5 29 1.95 1.9 Figure 4 Analysis of plasmid DNA purified with Lysate Direct PhyTip columns. Eight 16 hour grown bacteria culture carrying pcr4-topo plasmid were purified using Lysate Direct PhyTip columns. (A) Yield, concentration, A26/A28 ratio and A26/A23 ratio were measured using nanodrop and results are summarized in the table. (B) Agarose gel analysis of non-linearized plasmid (pcr4-topo ) purified using Lysate Direct PhyTip columns. Purified samples were run on a 2% agarose gel, stained with ethidium bromide. Lane 1-8 = Eight independent samples purified using Lysate Direct PhyTip columns,1 μl sample loaded. Lane 9 = 1 Kb Plus ladder (4 ng). Identical growth and purification conditions were used for each sample.
Figure 5: Sequencing Analysis with Lysate Direct PhyTip column purification method Fig 5A Fig 5B Figure 5 Sequencing Analysis with Lysate Direct PhyTip column purification method. Plasmid DNA purified with Lysate Direct PhyTip columns were sent out for sequencing on ABI 373xl instrument. Sequence was analyzed using Sequence Scanner verson 1. software and 784 continuous read length with 754 of the bases with QV >=2 was obtained. (A) Sequence peaks from 275 to 336 bases are shown. The sequence peaks are very sharp with minimal background noise peaks. (B) Sequence peaks from 544 to 66 bases. The peaks are still very sharp with slight increased background noise peaks which does not interfere with sequence read.
Figure 6: Transfection Efficiency Analysis with Lysate Direct PhyTip column purification method # of GFP positive cells 5 4 3 2 Transfection Efficiency 96 hours Reverse Transfection Mean Fluorescence in millions 2 15 5 DNA Quality 96 hours Reverse Transfection # of GFP positive cells 5 4 3 2 Transfection Efficiency 96 hours Forward Transfection Mean Fluorescence in millions 2 15 5 DNA Quality 96 hours Forward Transfection Figure 6 Transfection Efficiency Analysis with Lysate Direct PhyTip column purificatoin method. Plasmid DNA encoding GFP was purified using Lysate Direct PhyTip columns and then tested for transfection efficiency. 5 ng of plasmid DNA was transfected to COS7 cells using three different transfection reagents (Fugene 6, Fugene HD, and TransIT LT1). Transfections were carried out as per manufacturers suggested protocol. Ninety-six hours after transfection, GFP positive cells were counted and mean fluorescence calculated for each method using IncuCyte instrument.
Fig 4: Analysis of plasmid DNA purified with Lysate Direct PhyTip columns 2. Yield 25 Concentration 15. 2 ug 1. ng/ul 15 5. 5. 2.2 A26/A28 Ratio 2.2 A26/A23 Ratio A26/A28 Ratio 2. 1.8 1.6 1.4 1.2 A26/A23 Ratio 2. 1.8 1.6 1.4 1.2 1. 1. Fig 4: Eight 16 hour grown bacteria culture carrying pcr4-topo plasmid were purified using Lysate Direct PhyTip columns. Yield, concentration, A26/A28 ratio and A26/A23 ratio were measured using nanodrop.