Procedures

Materials

  • E. coli strain JM109 cells with plasmid grown overnight in LB media with 50 μg/mL ampicillin
  • Thermo Scientific GeneJET Plasmid Miniprep Kit. The composition of the solutions in this kit are unknown, however the manual cites reference 3 which uses:
    • Resuspension solution: GTE solution, sterile (50 mM glucose, 25 mM Tris·HCl, pH 8.0, 10 mM EDTA, 0.1 g/L bovine pancreatic RNase A)
    • Lysis solution: NaOH/SDS solution (0.2 M NaOH and 35 mM SDS)
    • Neutralization solution: 5 M potassium acetate solution (pH 4.8) with guanidinium chloride, unknown concentration
    • Wash solution: 0.3 M potassium acetate in 70% ethanol
    • Elution buffer: This is usually TE buffer: 10 mM Tris·HCl (pH 8.0), 1 mM EDTA
  • Spin columns (if spin columns are unavailable: 25:24:1 phenol:chloroform:isoamyl alcohol will be used)
  • 95% ethanol
  • 70% ethanol
  • TE buffer, pH 8.0, sterile (10 mM Tris·HCl, 1 mM EDTA, pH 8.0)
  • 1 kb Plus DNA reference ladder solution (New England Biolabs, N0550S) - formerly called 2-log ladder
  • DNA loading dye (Sigma, G2526)
  • Agarose
  • TBE buffer (8.9 mM Tris base, 89 mM boric acid, 2 mM EDTA)
  • DNA staining solution (0.5 μg/mL ethidium bromide in TBE buffer)
  • Set of pipettors and compatible sterile pipet tips
  • Ice and ice bucket
  • 1.5 mL microcentrifuge tubes
  • -20 °C frost-free freezer
  • Microcentrifuge
  • Vortexer
  • Thermo Scientific NanoDrop Onec Microvolume UV-Vis Spectrophotometer
  • Horizontal gel electrophoresis system with power supply
  • UVP BioSpectrum 500 Imaging System (configured for 302 nm transillumination)

 

Isolation of plasmid DNA

To save time, steps 1 and 2 will be performed for you.

1. LB medium (500 mL) containing the appropriate antibiotic was innoculated with a single bacterial colony. This was grown with shaking at 37 °C overnight (12-16 hours) in a 2 L flask while shaking at 200 rpm.

2. Culture (2 mL) was centrifuged for 2 minutes in a microcentrifuge at 7000 xg to pellet the bacteria. The supernatant was decanted into a labeled waste beaker.

The plasmid is known to be a low copy number plasmid (10-50 copies per cell). As a result, up to 10 ml of culture can be processed before the spin column reaches capacity (20 μg of dsDNA).

3. You will receive a pellet resulting from the prior steps. Write your initials on the cap do that you can locate it when you share the centrifuges. Wash away any remaining media by gently resuspending the pellet in 250 µl of Resuspension Solution. Centrifuge for 2 minutes in a microcentrifuge at 7000 xg. Remove the supernatant as completely as possible by tapping the tube into a liquid waste beaker.

4. Gently resuspend the pellet in 250 µl of Resuspension Solution by pipetting up and down. Poor resuspension of the pellet will significantly affect the yield of plasmid DNA. Read the Safety and Disposal instructions at the bottom of this page before handling the bacteria.

What's going on? The solution you are adding is 50 mM glucose, 25 mM Tris-HCl (pH 8.0), and 10 mM EDTA (pH 8.0). The glucose maintains external osmotic pressure so the cells don't burst before the next step. Tris buffer maintains physiological pH. EDTA inhibits endonuclease enzymes that degrade DNA by tightly binding all Mg2+ and Ca2+, ion cofactors found in DNA-degrading enzymes. RNase A degrades RNA after lysis. 

5. Add 250 μl Lysis Solution, mix by inverting the tube 4-6 times until the solution becomes viscous and slightly clear. Do not vortex to avoid shearing of chromosomal DNA. Do not incubate for more than 5 min to avoid denaturation of supercoiled plasmid DNA.

What's going on? The Lysis Solution contains SDS and NaOH. SDS detergent disrupts cell membranes and allows the NaOH to contact and denature both genomic and plasmid DNA. The SDS also denatures most proteins from the cell. This dissociates proteins that bind DNA. After SDS dissolves the cell membrane, the cell contents will partially neutralize the NaOH, reducing the pH to 11.5–12.3, depending on the cell volume. The hydrogen bonds of the DNA double helix denature above pH 10. When the genomic DNA denatures, it's long single strands become highly tangled and the material precipitates out of solution when the pH is dropped in the next step. In contrast, plasmid DNA renatures when the pH is dropped due to its small size. DNA is also hydrolytically unstable above pH 12 at room temperature. When the solution is incubated on ice, hydrolysis is not significant. If too little bacteria is used, the solution may be basic enough to degrade DNA.

6. Add 350 µL of the Neutralization Solution and mix immediately (to avoid localized precipitation of bacterial cell debris) and thoroughly by inverting the tube 4-6 times. It is important to mix thoroughly and gently after the addition of the Neutralization Solution. The neutralized bacterial lysate should become cloudy.

What's going on? The 5 M potassium acetate acidifies the solution to pH 4.8. This precipitates the sodium salts of SDS and the cell debris, including the genomic DNA. It is easy for the small circular plasmid DNA to re-nature, however it is almost impossible to properly anneal those huge genomic DNA strands. It is essential that mixing be complete. However, vortexing or other forms of harsh mixing, will shear the genomic DNA, resulting in short double stranded DNA fragments that will contaminate the solution, which currently contains plasmid DNA, RNA, protein, and small molecules. Since the SDS precipitates under these conditions, guanidinium is added to keep the proteins denatured.

7. Centrifuge for 5 minutes in a microcentrifuge at maximum speed to pellet the solid debris. If your instructor has specified that spin columns are unavailable in a News announcement, prepare the alternative procedure at this link. Otherwise, continue to the next step below.

8. Transfer the supernatant (~900 μl) to a GeneJET spin column by pipetting. Avoid disturbing or transferring the white precipitate.

9. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.

10. Add 500 μl of the Wash Solution to the GeneJET spin column. Centrifuge for 30-60 seconds and discard the flow-through. Place the column back into the same collection tube.

What's going on? Being less polar than water, ethanol disrupts the screening of charges by water. At 70% ethanol, the electrical attraction between phosphate groups and the cations (mostly K+) becomes strong enough to form stable ionic bonds and DNA precipitates as a neutral solid on the surface of the silica. All other material on the column flows through.

11. Repeat the wash procedure (step 9) using 500 μl of the Wash Solution.

12. Discard the flow-through and centrifuge the column with nothing in it for an additional 1 min to remove residual Wash Solution. This step is essential to avoid residual ethanol in plasmid preps.

13. Transfer the entire blue GeneJET spin column into a fresh 1.5 ml microcentrifuge tube. Add 50 μl of the Elution Buffer to the center of GeneJET spin column membrane to elute the plasmid DNA. Take care not to contact the membrane with the pipette tip. Incubate for 2 min at room temperature and centrifuge for 2 min with GeneJet spin column lid closed and 1.5-mL tube lid pointing towards the center of the centrifuge.

 

Characterization of Isolated DNA with Nanodrop spectrometer

If your instructor has specified that the Nanodrop will be unavailable (on the Main Page next to the link for this lab), prepare the alternative procedure at this link.

1. The first user should blank the instrument with 2 μl of Elution buffer. All class measurements can be made after a single blank measurement.

2. Load 2 μL of DNA sample onto the pedestal.

3. Record DNA concentration, A260/A280 ratio, and A260/A230 ratio. The reading should be in the instrument's linear absorbance range of 0.01 - 550 (0.5 - 27,500 ng/μL).

4. Clean the pedestal with DI water and a Kimwipe for the next user.

Tip: In practice, the minimal concentration that allows the reliable quantification of DNA is 0.05. A260 measurements below this value are generally unreliable.

Notes on claculations: The NanoDrop provides the results listed in step 3. In the report you will be asked to list A260, A280, and A230. You can calculcate these based on the relationship that concentration is 1.00 absorbance at A260 is 50 ng/μl of double straned DNA. After calculating A260, from this relationship, you can calculate the other abasorbances based on the ratios.

 

Agarose gel of isolated plasmid DNA

1. Your instructors have prepared a 0.7% agarose gel for visualizing contaminating RNA and genomic DNA. Prepare a sample by mixing 6 μL of your concentrated plasmid sample with 1.5 μL of 5X loading dye on a square of Parafilm.

2. Using a 20 μL tip, load this sample into a free well of the agarose gel. Record the position of the lane containing your sample. This sample will be run for 60 minutes at 90 Volts and, stained with ethidium bromide, and photographed via transillumination with 302 nm UV light. You will receive your results next week.

3. Give the rest of your concentrated DNA sample to your instructor or TA before leaving. You will need it next week!

 

SAFETY AND WASTE DISPOSAL

  • For bacteria spills, you must contact your instructor or TA for assistance. The instructor or TA will immediately cover twice the spill zone area with 10% freshly prepared bleach, cover with paper towels, and wipe-up after a 10 minute contact time. They will make certain that the spill has been completely contained and all materials that contacted the spill are disposed of in the Biohazardous Waste Bin.
     
  • Collect liquid waste in labeled beakers at your bench. When you are finished, collect your liquid waste in the Biohazardous Liquid Waste container found in the waste hood. Spray your waste beaker with freshly prepared 10% bleach, wait 10 minutes, then wash the beaker before leaving.
     
  • All gloves, pipette tips, tubes and other solids that have come in contact with the bacterial culture must be disposed of in the Biohazardous Solid Waste container on each bench and disposed of in the Biohazardous Waste Bin at the end of class.
     
  • Chloroform-phenol solution is toxic. The solution must remain in the fume hood. Students must keep tubes outside the hood closed as often as possible. After the supernatant has been removed, the chloroform solution must be disposed of in Chloroform Waste container in the fume hood. The used tubes should be disposed of open in the Solid Biohazardous Waster Container also in the hood.
     
  • The culture medium contains ampicillin. Students who have a penicillin allergy should work with their partner to make sure that they are not exposed.
     
  • Ethidium bromide is a suspect carcinogen. The Instructor and/or TA will handle materials contaminated with this chemical. Gloves, gels, and all other materials that contact ethidium bromide must go into the ethidium bromide solid waste bucket. Assume that the gel imager is contaminated and wear gloves.