Team:Hawaii/Large-Scale Preparation of Plasmid from E. coli

Large-Scale Preparation of Plasmid DNA
A protocol for the preparation of plasmid DNA from large cultures. Adapted from a protocol for a 500mL prep. Yields milligram quantities of reasonably clean crude DNA. To obtain highly purified plasmid DNA can be obtained by using CsCl/ethidium bromide equilibrium centrifugation (protocol found in Maniatis).

Also included are two assays for verification and quantification of plasmid DNA: UV wpectrometric quantification and restriction digest followed by gel electrophoresis.

Materials

 * LB medium containing selective agent
 * Plasmid bearing E. coli strain


 * GTE solution (50mM Glucose/10mM EDTA/25mM Tris-HCl, pH 8.0)
 * NaOH/SDS solution (0.2M NaOH/1.0%SDS)
 * 10mg/mL Rnase in Tris-HCl, pH 7.5
 * Isopropanol
 * 70% (v/v) ethanol
 * centrifuge
 * 2L baffled flask
 * 50mL centrifuge tubes
 * Water Bath at 55&deg;C

Procedure

 * 1) Innoculate 5mL LB containing selective agent with a colony of plasmid bearing E. coli. Grow at 37C with vigorous shaking over night.
 * 2) Inoculate 500mL LB containing selective agent with ~1mL of over night culture. Grow at 37C with vigorous shaking until OD600~4.0 is reached (saturation).
 * 3) *use baffled 2L flask
 * 4) *for this prep, we used a 300mL culture.
 * 5) Centrifuge 10 minutes at maximum 894 rcf (maximum rcf for our centrifuge), 4&deg;C. Use 50mL aliquots.
 * 6) *The protocol recommends using 6,000 x g.
 * 7) *The 300mL prep is divided into 6 50mL tubes.
 * 8) Combine 3 tubes by resuspending in 4mL of GTE solution. Incubate 10 minutes at room temperature.
 * 9) *For one variation (latter called Prep 1), add 50ug/mL Rnase solution (20uL).
 * 10) Add 10mL NaOH/SDS solution, mix (gently) by inverting 4 times. Incubate on ice for 10 minutes.
 * 11) *Solution should become homogeneous and clear. This prep was not clear, but proceeded with protocol.
 * 12) Add 7.5mL potassium acetate, mix (gently)by inverting 4 times. Incubate on ice for 10 minutes.
 * 13) *A white precipitate forms.
 * 14) Centrifuge for 15 minutes at 894 rcf, 4&deg;C.
 * 15) *Recommended to spin at 20,000 x g for 10 minutes.
 * 16) *Centrifuge until a good pellet forms. Some material will be floating, remove as much as possible with pipette tip.
 * 17) Decant supernatant to a new tube.
 * 18) *Do this step with a pipette and avoid the white precipitate.
 * 19) *For one variation (latter called Prep 2), add 50ug/mL Rnase solution (20uL).
 * 20) Add 0.6 volume of isopropanol. Mix by inversion, let stand 5-10 minutes at room temperature.
 * 21) *For a 21.5mL prep (total volume up to this point) add 12.9mL isopropanol.
 * 22) Centrifuge 15 minutes at 894 rcf at room temperature. Discard supernatant.
 * 23) *Recommended to spin at 15,000 x g.
 * 24) *Centrifuge until really good pellet forms. Avoid the pellet!
 * 25) Wash pellet with 2mL 70% ethanol.
 * 26) Centrifuge for 5 minutes at 894 rcf at room temperature. Aspirate ethanol.
 * 27) *Recommended to spin at 15,000 x g briefly.
 * 28) *Centrifuge until really good pellet forms.
 * 29) Dry pellet in the hood.
 * 30) *Recommended to dry the pellet under vacuum.
 * 31) Resuspend in 100uL TE, lightly vortex.
 * 32) *Recommended to store indefinitely at 4&deg;C (but does not specify if buffer is needed).
 * 33) Heat at 65&deg;C for 30 minutes.
 * 34) *The product was cloudy so taking extra purification step.
 * 35) Centrifuge 10 minutes at 894 rcf, room temperature.
 * 36) Aspirate clear liquid, avoiding pellet.
 * 37) Wash pellet with 100uL TE, centrifuge, aspirate and combine with product.
 * 38) Check the concentration of the plasmid using a spectrophotometer (need protcol).
 * 39) Verify presence of plasmid DNA by first using a restriction digest, followed by gel electrophoresis.

UV Spectroscopy for the Quantification of Plasmid DNA

 * used to asses purity and concentration of nucleic acids
 * A260 measurements are quantitative for relatively pure nucleic acid preps in microgram quantities
 * Cannot be used to discriminate between RNA and DNA
 * Ratio of A260/A280 indicates purity, as protein absorbs at 280nm.
 * A325 indicates particulates in solution or dirty cuvette
 * A230 for contaminants containing peptide bonds or aromatic moieties such as protein and phenol

Materials

 * 1x TE buffer
 * nanopure water
 * plasmid prep (for concentrated preps, need several dilutions)
 * pipetter (for 2uL quantity)
 * tips
 * nanodrop spectrophotometer
 * chem-wipes

Procedure

 * 1) Turn on computer, select spec icon, choose nucleic acids setting
 * 2) Pull up lever of spec (DON'T PULL with WIRE!), wash top and bottom with small amount of water.
 * 3) Blank with 2uL TE, click blank
 * 4) Load 2uL sample, click measure
 * 5) If results significant, can print. If not, repeat steps with a dilution of sample.

Materials

 * EcoRI
 * EcoRI buffer
 * BSA
 * nanopure water
 * pRL1383a (from prep 1 and prep 2)
 * heat block at 37&deg;C (later 65&deg;C)
 * 1.5 mL centrifuge tubes
 * agarose gel
 * gel apparatus
 * Ethidium Bromide
 * 6X loading buffer
 * Ladder

Procedure

 * 1) Turn on heat block.
 * 2) To the centrifuge tube, add 2uL nanopure water, 1uL EcoRI buffer, 1uL BSA, 5uL pRL1383a plasmid prep.
 * 3) Microcentrifuge until all contents are in solution at bottom of tube.
 * 4) Add 1uL EcoRI.
 * 5) Place on heat block for 1 hour.
 * 6) While waiting, prepare the gel.
 * 7) Microwave the gel in bottle in 30 second intervals, mixing in between until all of gel is melted.
 * 8) add Ethidium Bromide to gel.
 * 9) *There should be some already in the gel, adjust as needed.
 * 10) Put in well clip, pour gel after it is sufficiently cooled (does not hurt when you touch it).
 * 11) After 1 hour, increase heat on block to 65&deg;C and incubate digestion for 20 minutes.
 * 12) *Heat inactivation of EcoRI.
 * 13) Prepare samples for gel electrophoresis:
 * 14) Plan out wells, in addition to digestion, add circular plasmid as a control.
 * 15) Mix sample + loading buffer in 5:1 ratio.
 * 16) Load Ladder (NEB 2-log Tri-dye Ladder) and samples.
 * 17) Run gel at 95V for 45 minutes or until end of ladder reaches halfway down gel.
 * 18) Take some pictures.

Electrophoresis




Discussion

 * A large scale plasmid prep can be performed with this procedure.
 * From the gel electrophoresis results, is not apparent that the step in which the Rnase is added is important because a smear in the low kb region is found in both preps indicating a possible RNA contamination. We should add as the manual says after the GTE addition so that the experiment can follow this manual as closely as possible.
 * To improve the RNA digestion, we will adjust the protocol by incubating the RNase for 30 minutes at 55&deg;C.
 * The plasmid prep from 7/3/08 still contained RNA, so as per Dr. Callahan's advice, we will add the rnase in the later step and incubate for 1.5 hours at 55&deg;C.
 * From the gel electrophoresis results, we can conclude that it is necessary to linearize the plasmid DNA before a gel is run. It is also apparent that levels of Ethidium Bromide should be added so that sufficient staining is achieved.
 * We should use a 1% gel as opposed to a 2% gel. This is probably why our DNA did not run.