Plasmid Purification

Conjugation lab:

You will perform a multi-step examination of  bacterial conjugation, which is the transfer of plasmid DNA from one cell to another. This experiment will be spread over six lab days and will include these steps:

Plasmid purification is also part of the pGLO lab.

In this lab procedure, you'll purify plasmid DNA from bacterial cultures. You will use this technique two different times in Bio 6B, for the plasmids pARO180 and pGLO. When you ran your total nucleic acid gel, you probably used a small sample of purified plasmid as a size marker. Now you can purify your own, and later analyze it with restriction digests and a gel.

Background

Purifying specific types of macromolecules is a fundamental technique in molecular biology and biochemistry. So far, you have used lysates for proteins (in the SDS-PAGE lab) and for nucleic acid (for the total nucleic acid gel of the conjugation lab). Both those techniques gave you the macromolecules you were looking for, but not in a pure form; you ended up using specific stains for proteins or nucleic acids on your gel so you could see your desired results.

The plasmid purification technique used in this lab is much more specific. Not only will it eliminate proteins and other cell components from your plasmid DNA, it will also eliminate the chromosomal DNA so you end up with a very pure sample of plasmid DNA. This is important for many downstream uses of plasmids, such as restriction digests, sequencing, and transformation.

This procedure described on this page uses the Promega Wizard Miniprep kit. "Miniprep" means that it starts with a single microcentrifuge tube of bacterial culture. Several companies make similar kits. They don't tell us exactly what's in the solutions, but it's based on a classic technique known as alkaline lysis.

Alkaline lysis and plasmid purification

The detailed steps are described in the protocol section below, but it's worth taking a moment to consider what these steps are supposed to do.

Culture and pellet
You grow a dense culture of bacteria and spin it down into a pellet, concentrating the cells.
Resuspend
The cell resuspension solution gently resuspends the cell pellet, so you can lyse the cells quickly and evenly in the next step.
Lyse
"Lyse" means to split the cells open (to cause lysis). The lysis buffer contains SDS, which solubilizes both membranes and proteins. It also contains a strong base such as NaOH. The alkaline pH disrupts the hydrogen bonding that holds the two strands of DNA together, so the DNA will be single-stranded.
Neutralize
The neutralization buffer restores pH to near neutral, and contains some ions. The neutral pH allows plasmid DNA to renature, returning to its original double-stranded state. However, the much-larger chromosomal DNA can't renature fully. Also, in these conditions, the SDS and denatured proteins tend to aggregate and precipitate; the partially denatured DNA precipitates along with them and ends up in the pellet.
Column purify
After a spin, the neutralized lysate contains plasmid DNA. This solution is passed through a mini-column inside a microcentrifuge tube. The column contains a matrix typically made of silicon dioxide (glass) particles. The solution contains chaotropic salts, which weaken the hydrogen bonding in the water, and making the DNA less soluble. Under these conditions, the DNA binds to the glass matrix. The chaotropic salts are washed away with a buffer that contains alcohol; this wash buffer dissolves the salts but not the DNA. Finally the alcohol is removed and the pure DNA can be eluted, or dissolved off the glass matrix of the column, in pure water or TE buffer. In the end, the column step concentrates the DNA while purifying it, since the elution volume is smaller than the original volume of the lysate.

Related techniques

The purification step in the plasmid purification protocol is a form of column chromatography. The essence of column chromatography is that there are two phases: a stationary phase (the glass matrix inside the column) and a mobile phase (the various buffers that you pass through the column). If molecules have greater affinity for the solid phase than for the liquid phase, they bind to the column. If they have greater affinity for the liquid phase, they are eluted (washed away) from the column. In plasmid DNA purification, you reduce the affinity of the DNA for the aqueous liquid phase two different ways: first, you use chaotropic salts, which weaken hydrogen bonding and decrease the solubility of DNA. Next, you use a wash buffer containing alcohol, which also decreases the solubility of DNA. Under these conditions, the DNA binds to the column. Finally you elute the DNA using pure water, which readily dissolves DNA.

Column chromatography is similar to the liquid-liquid extraction that you performed with chloroform. In that case, some cell components had a higher affinity for chloroform, and they were pulled away from the aqueous phase. Nucleic acids, which are highly water-soluble, remain in the aqueous (water) phase.

The plasmid purification method is also similar to hydrophobic interaction chromatography (HIC), which you will use as a protein purification technique in the pGLO lab. In HIC, you will also use chaotropic salts to weaken the interaction between water and protein, so the protein wil bind to the stationary phase in the column. Unlike the plasmid DNA column, the HIC column is hydrophobic, and the proteins bind by hydrophobic interactions. In both the DNA and protein columns, you can elute your macromolecules by washing away the chaotropic salts and dissolving the macromolecules in water.

Starting the cultures

You'll start the cultures one lab period and let them grow until the next. You'll purify four different plasmids, so you'll need to start four different liquid cultures:

  • E. coli HB101 containing the plasmid pARO180. Start this culture from the amp+strep plate from your conjugation experiment.
  • E. coli HB101 containing the unmutated version of the plasmid pGLO, which produces green colonies.
  • E. coli HB101 containing the mutated version of the plasmid pGLO that produces blue colonies.
  • E. coli HB101 containing the mutated version of the plasmid pGLO that produces non-fluorescent colonies.

Use a liquid culture in LB broth, because liquid cultures are the best way to produce large numbers of cells, and LB provides all the nutrients needed for growth. Both cultures should contain ampicillin, because both plasmids contain the gene for β-lactamase, the ampicillin resistance gene. It's always possible that some cells will lose their plasmids, simply by chance. If you don't add the antibiotic, the no-plasmid cells, having less DNA to copy, will grow faster, and may eventually dominate the culture (especially if you grow the culture for more than 24 hours). Adding the antibiotic ensures that only cells containing the plasmid can grow.

Calculating the amount of ampicillin

Add ampicillin to your culture tube to a final concentration of 100 μg/ml. The concentration of the stock solution might be 50 mg/ml, but you should check. The volume of LB broth in the tube varies; often it's 4 ml. If you're not sure, you can get an empty culture tube and pipet some water in there, 1 ml at a time, until the volume matches the LB tube you are using.

Use C1V1=C2V2 to calculate the appropriate amount of antibiotic stock solution to add. If you think this calculation requires a calculator, you might want to look at the amazing milli x micro trick on the Calculations page. Also, once you do the calculation, you should realize that you're only adding a small amount of antibiotic solution; if you have 4 ml LB and you add a tiny amount of amp, the final volume will still be approximately 4 ml.

How to inoculate the cultures

To start a liquid culture from a plate, use a sterilized metal loop to scrape a tiny bit of a colony from the plate, then twirl the loop in the liquid LB. If you can see any cells transferred into the liquid, it's plenty. Don't scrape a large blob into your liquid; the culture will become overgrown.

Plasmid purification protocol

Materials

  • Your tubes of liquid bacterial culture containing pARO180 and pGLO plasmids (including regular pGLO and mutant pGLO). If you grew your cultures in LB without arabinose, they won't be fluorescent.
  • Test tube rack to hold the culture tubes
  • 1 empty glass culture tube (for waste)
  • One microcentrifuge tube (1.5 ml) for each sample and a rack to hold them
  • Cell Resuspension Solution
  • Cell Lysis (alkaline detergent) Solution. Before using, make sure there is no precipitate in this tube. If there is, gently warm the tube and mix (but don't shake it up) until no precipitate is visible.
  • Neutralization Solution
  • Alkaline Protease Solution
  • Spin Columns (“Wizard® SV Minicolumn”). One for each sample.
  • Collection tubes. One for each sample.
  • Column Wash Solution. This solution comes in a bottle with the kit, but it ethanol must be added to it before use. Don't use a bottle directly out of the kit if the ethanol hasn't been added yet.
  • Sterile water

Method

The instructions below describe the steps for a single culture; you should be doing two samples in parallel.

  1. Pipet 1.5 ml of bacterial culture into a microcentrifuge tube. Pipet the thick gob of stuff at the bottom of the culture tube to get as many cells as possible.
  2. Spin at 13,000 rpm (high speed) for 5 minutes to pellet cells.
  3. Pipet off the supernatant into an empty glass culture tube. Don't put it in your beaker with your waste tips. Do not combine liquid waste and solid waste.
  4. Add 250 µl of Cell Resuspension Solution to the microcentrifuge tube containing the bacterial pellet and completely resuspend the cell pellet by pipetting. Be sure the whole pellet gets resuspended – don’t leave any chunks. (You can mix vigorously at this point because the DNA is still inside the cells.)
  5. Add 250 µl of Cell Lysis Solution to the resuspended pellet and mix by inverting the tube 4 times. Incubate at room temperature for 30 seconds. Mix gently at this point because the cells have lysed and the DNA is free in solution and has been denatured to a single-stranded form.  The DNA molecules can be damaged by excessive pipetting or mixing.
  6. Add 10 µl Alkaline Protease Solution and mix by inverting the tube 4 times. (Alkaline protease is a protein-digesting enzyme that works in the alkaline conditions of the cell lysis solution. It destroys enzymes that might damage the DNA.) Incubate 1 minute at room temperature.
  7. Add 350 µl of Neutralization Solution and mix by inverting the tube 4 times. This neutralizes the highly alkaline pH of the lysis solution; if DNA is left too long in the lysis solution, it will be damaged.
  8. Centrifuge the lysate for 10 min at 13,000 rpm. When the spin is done, you’ll have a cleared lysate. Meanwhile, get your Spin Column ready.
  9. Insert Spin Column into Collection Tube. Label both column and tube. (Fingertips courtesy of Jessica Gutierrez.)

Plasmid column 400px

  1. Get your lysate out of the centrifuge; you should now see a cleared lysate (the supernatant) and a white precipitate. Transfer 800 µl of the supernatant into the Spin Column. Be sure not to transfer any of the pelleted precipitate.
  2. Centrifuge the Spin Column at 13,000 rpm for 1 minute. The lysate will go through the Spin Column, but the DNA sticks to the column. You can spin the tube without a lid, but balance the centrifuge carefully. Make sure all the liquid goes through the column for this and for subsequent spin column steps. The exact timing of the spin isn’t important; the important thing is getting the liquid though the column.
  3. The liquid that went through the Spin Column is waste. Your DNA should now be bound to the column. Use a glass culture tube as a waste container for the liquid; you can either pour it or pipet it from the collection tube into the waste tube.
  4. Add 750 µl Column Wash Solution (which was previously diluted with ethanol) to Spin Column.
  5. Centrifuge at 13,000 rpm for 1 minute. The solution goes through, but the DNA remains stuck to the Spin Column. Pour off the wash solution from the Collection Tube. The wash solution that ends up in the Collection Tube is waste; transfer it to your waste tube before doing the next step.
  6. Add another 250µl Column Wash Solution to Spin Column and spin for 5 minutes. This long spin is important to eliminate all the ethanol from the wash solution. Be sure all the liquid passes through the column. If some wash solution, which contains ethanol, remains in the column, you will not recover all your DNA and you not be able to load your DNA onto a gel later. If there is ethanol in your  final DNA solution, the sample will have low density and will tend to float when you load the gel. The wash solution that ends up in the Collection Tube is waste.
  7. Transfer Spin Column to a new, sterile 1.5 ml microcentrifuge tube. Be sure there is no more wash solution in or on the spin column; it should be dry. If the lid of the microcentrifuge tube closes on top of the spin column, close it. If it doesn't close, pull or cut the lid off the tube and save it for later.
  8. Add 100 µl sterile water to the spin column. The water will elute the DNA, dissolving it off the column. Be sure to use sterile water from a glass bottle, not deionized water from the tap (which isn't sterile).
  9. Centrifuge for 1 minute at 13,000 rpm. Be sure all the liquid goes through. The liquid that goes through the column contains your plasmid DNA. Throw away the spin column, close the tube containing the eluted DNA, and save it in the freezer until next lab period.

Before you leave: sample storage and waste disposal

You should have a tube of plasmid DNA from each of your samples (pARO180, pGLO, pGLO Blue mutant, pGLO non-fluorescent mutant) in a rack with everyone else’s DNA in the freezer.

Glass culture tubes with lids: peel off the tape labels and put the tubes in a tube rack in the dirty-glassware tub on the cart. Don't dump out the liquid; it will be sterilized and disposed of later. Don’t set the tubes on their sides. Don’t leave the tubes in a beaker. Leave them in a rack.

Microcentrifuge tubes and pipet tips go in the biohazard trash. Gloves, paper towels, etc. go in the regular trash.

Beakers used for waste tips: dump the tips into the biohazard trash and put the beaker into the dirty-glassware tub on the cart (if there’s not a tub, check with the instructor).

Review

Terms and concepts

  • Alkaline lysis
  • Chaotropic salts
  • Column chromatography
  • Denaturing DNA
  • Lysis (noun, the process of breaking cells open); lyse (verb; to break open the cells); lysate (noun, the material released from cells during lysis).
  • Solid phase vs. liquid phase

Review questions

  1. Why was it important to use antibiotics in the cultures?
  2. How do the cells get lysed in this procedure?
  3. How are proteins eliminated?
  4. How is chromosomal DNA eliminated?
  5. Why does plasmid DNA bind to the stationary phase in the column at one point in this experiment? Why is the DNA released into the aqueous mobile phase (eluted) from the column at another point in the experiment?
  6. Compare and contrast the total nucleic acid extraction procedure with the plasmid purification procedure. Why did we use each of these methods? Why do they work?
  7. Why is it so important to be sure you remove all the wash buffer from the column before the elution step?
  8. You'll use your purified plasmids from today's procedure for restriction digests, but you didn't do that with the DNA from the total nucleic acid procedure. Why?

References

Plasmid purification

The Basics: How Alkaline Lysis Works by Nick Oswald on Bitesize Bio. Offering a clear and simple overview of the technique, this is the best thing to read if you want to undersand the technique. Other relevant articles on the same site include How DNA Extraction Kits Work in the Lab and Plasmid vs. Genomic DNA Extraction: The Difference by Suzanne Kennedy.

DNA Purification: The basics of DNA isolation, plasmid growth and DNA quantitation from Promega, the company that produces the kit that we use. A very detailed guide to the general principles involved as well as the specific products.

Promega Wizard Plasmid Miniprep Instructions from Promega. Detailed manual, including troubleshooting.

Videos

How to Harvest Plasmid DNA with Minipreps. A technique video from Synthetic Biology One.

How To Perform a Plasmid Miniprep from Bio-Rad Explorer. 13 minutes.

Chaotropic effects

Both plasmid DNA purification and hydrophobic interaction chromatography (HIC) for proteins make use of chaotropic salts. The mechanisms by which chaotropic salts make macromolecules bind to the solid phase in a column make an interesting and somewhat controversial topic, which goes way beyond Bio 6B. If you google it, you'll find two kinds of explanations: those that are simple and clear, and those that are true. I don't think you can have both. If you're a chemistry minded-person, you might be interested in this:

Water structure and chaotropicity: their uses, abuses and biological implications. Philip Ball and John Hallsworth, 2012. Physical Chemistry Chemical Physics.

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