pGLO Plasmid Isolation

BACKGROUND

As we have demonstrated , it is relatively simple to insert a plasmid into bacterial cells. Likewise, it is relatively simple to remove a plasmid from bacterial cells. The development of these two protocols, along with the discovery of restriction enzymes, in the early 1970s led to the birth of the molecular biology revolution. By isolating plasmids, modifying their genetic content in vitro, and reinserting these altered plasmids into bacteria, researchers could create genetically modified organisms. The rest, as they say, is history.

Traditionally, methods of isolating plasmid DNA have relied on the small size of the plasmids relative to the chromosomal DNA. Centrifugation through a CsCl gradient was used originally to separate the two classes of DNA. This method, though, is time-consuming and expensive. Current protocols usually include a step that results in the co-precipitation of the large chromosomal DNA with the cell membrane. The plasmid DNA then is isolated through affinity chromotography. Such protocols are very rapid, inexpensive, and result in a pure preparation of plasmid DNA suitable for further experiments.


GOALSA

Learn the basic technique of plasmid isolation
Prepare a high quality preparation of pGLO for future use

MATERIALS

Escherichia coli strain K12 transformed with pGLO
Terrific broth + ampicillin

1.2% tryptone
0.5% yeast extract
0.05% NaCl
2.5mM KCl
10mM MgCl2
50 ug/ml ampicillin
pH 7.0

Cell Resuspension Buffer (Bio-Rad)
Cell Lysis Solution (Bio-Rad)
Neutralization Solution (Bio-Rad)
DNA Binding Matrix (Bio-Rad)
Wash Buffer (Bio-Rad)
Deionized water

PROTOCOL (adapted from Bio-Rad)

Night before lab: Using a steril loop, pick a single colony transformed with pGLO from your plate and transfer it to a tube containing 5ml Terrific broth. Place this tube in the 37oC shaking incubator.

1. Transfer 1.5 ml of overnight culture to a microcentrifuge tube.
2. Pellet cells by centrifuging at 12,000xg for 30 seconds. Pipet off and discard the supernatant. Add another 1.5 ml of overnight culture and repeat.
3. Add 200ul Cell Resuspension Buffer and resuspend the pellet by vortexing or pipetting gently.
4. Add 250ul Cell Lysis Solution and mix by gently inverting the tube several times. This solution usually consists of NaOH and the detergent SDS. The solution should become viscous and clear as a result of detergent lysis of the lipid bilayer of the bacterial cells.
5. Add 250ul Neutralization Solution and mix by inverting the tube several times. This solution, which generally contains potassium acetate and glacial acetic acid, causes the SDS and lipid bilayer to precipitate. The large chromosomal DNA also gets trapped in the precipitate.
6. Centrifuge the tube for 5 minutes at 12,000xg to pellet the SDS and cellular debris.
7. Transfer the supernatant to a spin filter inserted into a 2ml micrcentrifuge tube.
8. Add 200ul of thoroughly resuspended DNA binding matrix to the spin filter and pipet up and down to mix. The DNA binding matrix consists of silicon dioxide obtained from the exoskeletons of diatoms. This material has a high affinity for DNA and is very easy to handle. Glass micro-beads also can be used.
9. Centrifuge for 30 seconds at 12,000xg.
10. Remove and save the spin filter from the tube and discard the filtrate in the tube. Replace the spin filter.
11. Add 500ul of Wash Solution to the spin filter. Centrifuge for 30 seconds at 12,000xg.
12. Remove the spin filter from the tube, discard the filtrate and replace the spin filter.
13. Add 500ul of Wash Solution and centrifuge for 2 minuites at 12,000xg. The Wash Solution is 80% ethanol. This step serves to remove salts and other contaminants from the DNA.
14. Remove the spin filter and place it into a clean 1.5ml microcentrifuge tube. Add 100ul of water to the tube and centrifuge for 1 minute at 12,000xg to elute the DNA. The filtrate in this step is your purified plasmid DNA.
15. Store your plasmid DNA in the freezer. The DNA should be at a concentration of roughly 250 ng/ul.

At this stage, it often is desirable to measure the amount of DNA present in the sample and purity of the DNA. This measurement can be achieved with a spectrophotometer. Nucleic acid absorbs light maximally at 260nm. Therefore, by measuring the OD260 of a sample, one can calculate the concentration of nucleic acid in that sample. Generally, the formula used is OD260 x 50 = ug/ml of DNA. Furthermore, the OD260/OD280 ratio of pure DNA should be 1.8 - 2.0. A ratio below 1.8 indicates that contaminating materials (usually proteins) are present in the sample.



QUESTIONS

What role do the various steps play in this experiment?
Why were the bacteria grown overnight in medium containing ampicillin?