Gayatri Mehta Plasmid DNA Isolation-Midiprep Aim: To isolate plasmid DNA from bacteria using Microwave Method (mini

prep) and kit based midiprep method (Promega) Approach Theory A plasmid preparation is a method used to extract and purify plasmid DNA. Many methods have been developed to purify plasmid DNA from bacteria. These methods invariably involve three steps:

Growth of the bacterial culture Harvesting and lysis of the bacteria Purification of plasmid DNA

Plasmids are almost always purified from liquid bacteria cultures, usually E. coli, which have been transformed and isolated. Virtually all plasmid vectors in common use encode one or more antibiotic resistance genes as a selectable marker (Ex :kanamycin, Ampicillin), which allows bacteria that have been successfully transformed to multiply uninhibited. Bacteria are grown under favourable conditions. When bacteria are lysed under alkaline conditions both DNA and proteins are precipitated. Some scientists reduce the concentration of NaOH used to 0.1M in order to reduce the occurrence of ssDNA. After the addition of acetate-containing neutralization buffer the large and less supercoiled chromosomal DNA and proteins precipitate, but the small bacterial DNA plasmids can renature and stay in solution. Kits following this alkaline lysis protocol are available from varying manufacturers to purify plasmid DNA, which are named by size of bacterial culture and corresponding plasmid yield. In increasing order, these are the miniprep, midiprep, maxiprep, megaprep, and gigaprep. The plasmid DNA yield will vary depending on the plasmid copy number, type and size, the bacterial strain, the growth conditions, and the kit. Minipreparation of plasmid DNA is a rapid, small-scale isolation of plasmid DNA from bacteria. It is based on the alkaline lysis method invented by the researchers Birnboim and Doly in 1979. The extracted plasmid DNA resulting from performing a miniprep is itself often called a "miniprep". Minipreps are used in the process of molecular cloning to analyze bacterial clones. A typical plasmid DNA yield of a miniprep is 20 to 30 g depending on the cell strain.

MIDIPREP- The PureYield Plasmid Midiprep System (Promega Corporation)

As research moves from DNA sequencing to analysis of gene function, the need has increased for rapid methods by which to isolate large quantities of high-quality plasmid DNA. The PureYield Plasmid Midiprep System is designed to isolate high-quality plasmid DNA for use

Gayatri Mehta in eukaryotic transfection and in vitro expression experiments. The system provides a rapid method for purification using a silica-membrane column. Plasmid DNA can be purified in less than 45 minutes, greatly reducing the time spent on purification compared to silica resin or other membrane column methods. The PureYield Plasmid Midiprep System also incorporates a unique Endotoxin Removal Wash, designed to remove substantial amounts of protein, RNA and endotoxin contaminants from purified plasmid DNA, improving the robustness of sensitive applications such as eukaryotic transfection, in vitro transcription and coupled in vitro transcription/translation. Purification is achieved without isopropanol precipitation of purified plasmid DNA or extensive centrifugation, providing rapid purification as well as a high concentration of pure plasmid DNA from a single method. The PureYield Plasmid Midiprep System is designed to purify 100200g of plasmid DNA with an A260/A280 >1.7 from a 50100ml overnight culture of bacteria, transformed with a high copy number plasmid, with a total biomass (O.D.600 of culture volume of culture) of 100200. Larger amounts of biomass (e.g., up to 250ml of bacterial culture) can be processed with additional volumes of solutions. Requirements Bacterial culture: A. For midiprep 16-18 hour old broth culture of E.coli containing pRSET A vector grown in 50mL of LB broth containing 50g/mL of ampicillin. B. For miniprep Using Microwave Method 16-18 hour old broth culture of E.coli containing pRSET A vector grown in 5mL of LB broth containing 5g/mL of ampicillin. A. For midiprep Materials provided in the kit Cell Resuspension Solution (CRA) 12ml Cell Lysis Solution (CLA) 12ml Neutralization Solution (NSB) 20ml Endotoxin Removal Wash 15ml Column Wash 33ml Nuclease-Free Water 13ml PureYield Clearing Columns 4 nos PureYield Binding Columns 4 nos Storage Conditions: Store all system components at room temperature (2225C).

Gayatri Mehta

Materials to be supplied by the user Isopropanol Ethanol, 95% Luria Bertini Broth 50g/mL ampicillin stock Sterile assay tubes Sterile 100mL flask Tabletop Centrifuge 50ml Disposable Plastic Screw-Cap Tubes (E.G., Corning Or Falcon Brand) High-Speed Centrifuge Capable of At Least 15,000 G And Appropriate Tubes Vacuum Pump, Single- or Double-Stage, Producing Pressure Of Approximately 650mm Hg (25.6 Inches Hg) Vacuum Manifold

B. For miniprep Using Microwave Method Requirements Chemicals Luria Bertini Broth 5g/mL ampicillin stock Isopropanol STET 3 M Sodium acetate, pH 5.2 RNase 20g/ml TE, pH 8.0

Glassware Sterile Microcentrifuge Tubes Sterile Peteridish

Miscellaneous Tabletop Centrifuge Ice Microwave

A. For midiprep Preparation of solutions

Gayatri Mehta Before lysing cells and purifying DNA, prepare Endotoxin Removal Wash by adding isopropanol, and Column Wash by adding ethanol. Cap tightly after additions. The solutions are prepared as given below: Endotoxin removal wash 4 preps system (Cat.# A2490): Add 10ml of isopropanol to the Endotoxin Removal Wash bottle. Column wash 4 preps system (Cat.# A2490): Add 55ml of 95% ethanol to the Column Wash bottle. B. For miniprep Using Microwave Method Preparation of solutions STET 0.1M NaCl 10 mM Tris.Cl (pH 8) 1 mM EDTA (pH 8) 5% Triton X-100

Take 200 l of 5 M NaCl, 100 l of 1M Tris.Cl (pH 8), 20 l of 0.5M EDTA (pH 8), 500 l of the Triton X-100. Add 9.36 ml of sterile distilled water to make total volume to 10 ml. Lysozyme Weigh 10 mg of lysozyme and dissolve it in 1 ml of sterile distilled water. TE, pH 8.0 Sterile TE buffer : 10mM Tris Cl pH 8 1mM EDTA pH 8

For 10mM TrisCl solution 1ml of 1 M TE stock solution was diluted to 100ml with sterile distilled water. This was autoclaved and stored at room temperature RNase 20g/ml 3 M Sodium acetate, pH 5.2 A. For midiprep

Gayatri Mehta Procedure Standard DNA purification protocol Prepare Lysate Bacterial culture volume 50-100 ml 10 minutes 3 ml Bacterial culture volume 101-250 ml 10 minutes 6 ml 6 ml

Pellet cells at 5,000g Suspend pellet in Cell Resuspension Solution. Add Cell Lysis Solution. 3 ml Invert 3-5 times to mix. Incubate 3 minutes at room temperature. Add Neutralization Solution. 5 ml Invert 5-10 times to mix. Centrifuge lysate at 15,000 g at room temperature. DNA purification

10 ml

1. Assemble a column stack by placing a blue PureYield TM Clearing Column on top of a white PureYield TM Binding Column. Place the column stack onto a vaccum manifold. 2. Carefully pour supernatant into column stack. Apply vaccum, continuing until all liquid has passed through both the clearing and binding columns. 3. Slowly release the vaccum from the filtration device. Remove the blue clearing column, leaving the binding column on the manifold. NOTE: If the binding membrane has been dislodged from the bottom of the column, tap it back into place using a sterile pipette tip. Wash Add 5.0ml of Endotoxin Removal Wash to the binding column, and allow the vaccum to pull the solution through the binding column. Add 20ml of Column Wash Solution to the binding column, and allow the vaccum to pull the solution through the binding column. Dry the membrane by applying a vaccum for 30-60seconds. Repeat if the tops of the DNA binding membranes appear wet or there is detectable ethanol odor. Remove the binding column from the vaccum manifold, and tap it on a paper towel to remove excess ethanol.

4. 5. 6. 7.

Elute by Vacuum 8. Place a 1.5ml microcentrifuge tube into the base of the EluatorTM Vaccum Elution Device (Cat. #A1071), securing the tube cap.

Gayatri Mehta 9. Assemble the EluatorTM Vaccum Elution Device, and insert the DNA binding column into the device, making sure that the column is fully seated on the collar. 10. Place the elution device assembly, including the binding column, onto a vaccum manifold. 11. Add 400-600l of Nuclease-Free Water to the DNA binding membrane in the binding column. Wait for 1 minute. Apply maximum vaccum for 1 minute or until all liquid has passed through the column. 12. Remove the microcentrifuge tube and save for DNA quantitation and gel analysis. Elute by Centrifugation 13. Place the binding column into a new 50ml disposable plastic tube. 14. Add 600l of Nuclease Free Water to the DNA binding column. Wait for 1 minute. Centrifuge the binding column at 1,500-2,000 g for 5 minutes using a swinging bucket rotor, and collect the filterate. NOTE: Do not cap the 50ml tube during centrifugation.

B. For miniprep Using Microwave Method Procedure 1. Single isolated colonies grown on LB ampicillin agar plates were picked up and inoculated into 5ml of pre-warmed LB ampicillin broth and incubated in an orbital shaker at 37C, overnight to make small scale plasmid preparations. 2. An 850ml aliquot from the overnight grown culture was mixed with 150ml of glycerol and stored at 4C. 3. The remaining cell culture was centrifuged at 900 x g for 5 minutes at 4C. 4. The supernatant was aspirated leaving the pellet as dry as possible and then put on ice. Plasmid isolation was done with a method adapted from Hultner & Cleaver (1994). 5. The pellet was resuspended in 400 ml ice-cold STET by vortexing and allowed to stand for 10 minutes at 37C. 6. The microfuge tubes were then heated in a microwave for 18 sec and the tubes were quench cooled on ice for 5 minutes followed by centrifugation at 1300 x g, 15 minutes at 4C. 7. The cell debris and genomic DNA were removed with a toothpick. 8. The DNA was precipitated with equal volume of isopropanol and 1/10th volume of sodium acetate and incubated at 70C/2 hours or overnight. 9. The pellet obtained after centrifugation at 1300 x g /15 minutes/4C was washed using 70% ethanol, dried and resuspended in 30-40 ml of autoclaved distilled water containing RNase (200mg/ml) and incubated at 37C/ hour.

Gayatri Mehta Preparations for agarose gel electrophoresis (for Method A and B) 1. 1% agarose: Dissolve 0.5 g of agarose in 50 ml of 1X TAE buffer and digest using microwave. Add 2 l of ethidium bromide to it and cast the gel. 2. 1X TAE buffer: Dilute 20 ml of 50 X TAE buffer to 1000 ml with distilled water. For 50 X TAE: Tris base: 242 g Glacial acetic acid: 57.1 ml 0.5 M EDTA: 100 ml Make up the volume to 1000 ml with distilled water. The pH should be 8.0. 3. Ethidium bromide (EtBr): Dissolve 0.01 g of EtBr and dissolve in 2ml of distilled water. 4. 6X gel loading dye: Take 3.44 ml of available glycerol stock (87%) in a falcon tube. To it add 250 l of 10% bromophenol blue and 250 l of 10% xylene cyanol. Make the final volume to 10 ml with sterile distilled water. Store at 4oC. PROCEDURE Load the plasmid preps obtained from the three methods on 1% agarose gel casted in the gel electrophoresis apparatus as follow

Gayatri Mehta Observation

Fig: 1% agarose gel containing minipreparation and midipreparation of Plasmid DNA

Lane Number 1 2 3 4

Name

Hind III/ EcoR1 ladder (Fermentas) Midi-Prep Elute 1 pRset A Microwave Prep pRset B Microwave Prep pRset A

Results Interpretation Conclusion