Plasmid DNA purification from transformed bacterial colonies

Background:
There are a number of methods for DNA purification, all of which are carried out by some variation of basic procedures: lysis/rupture of ‘host’ cells and separation of liberated DNA from other cellular components in a form which is useable for intended downstream applications.

Over the years, the methodology for DNA purification has evolved towards procedures that are faster, easier, and more convenient than earlier methods, and involve minimal use of toxic organic chemicals (such as phenol and chloroform). In common use now are silica resins pre-packed into chromatography columns, combined with buffer systems that are designed to allow for specific binding of nucleic acids to the resin. Larger cellular ‘contaminants’ (macromolecules) are typically precipitated out and removed, either by centrifugation or filtration, from the ruptured cell mixture prior to the mixture’s application to the silica column. Subsequently, the mixture of nucleic acid plus smaller contaminants (metabolites, peptides, etc.) is applied to the column under conditions that favor nucleic acid binding. One or more ‘wash’ steps with the appropriate buffer(s) remove weakly bound contaminants, after which pure nucleic acid is eluted from the resin. (See “workflow” diagram, next page.)

Insert schematic

In this protocol, you will purify your plasmid DNA from other bacterial cell components in the lysates (subcellular preparations) of the host bacteria. Protocol is to be performed for two replicate samples (colonies) of transformed bacteria.

Reagents
Each team will need a bucket of ice. At each station you should have:

-Resuspension buffer (P1), containing RNase A and “LyseBlue” pH indicator, see below;
-Alkaline lysis buffer (P2);
-Neutralization buffer (P3) (keep on ice);
-Equilibration buffer (QBT) for Midi tip-100 silica-DEAE resin;
-Wash buffer (QC)
-Elution buffer (QF)
-Isopropanol;
-70% ethanol
(all solutions at room temperature, except for P3)

Other components of the system:
• Silica columns, syringe filters
Tubes for collecting reagent flow through (waste)
——————————————————————–

1. Harvest bacteria with your plasmid: Retrieve your two flasks of bacteria; if growth was successful, the media will be quite “cloudy”. Using sterile serological pipettes, transfer 14 ml of cell suspension from each flask to a labeled high- speed centrifuge tube. Eyeball your two tubes and make sure they’re at the same height; if not, make the necessary adjustments. Proper balance is important when centrifuging, especially at the higher speeds.

Spin the bacteria in the high-speed centrifuge (room 408) for 15 minutes at 6000 x g at 4oC. (We will likely spin the entire class’s samples together.) Carefully decant into designated container with bleach.

2. Resuspend the bacterial cell pellet: First, ‘shake’ or vortex your aliquot of P1
Resuspension buffer immediately before first use. (This resuspends the particles of “LyseBlue” pH indicator.) Then add 4 mL of P1 buffer to each of your bacterial pellets. Cap the tubes and vortex the mixture at high speed until there are no cell clumps remaining. It is important to completely resuspend the bacterial cells at this stage. Note that the bacteria are still intact at this point; you have not yet lysed them.

3. Lyse the host bacterial cells: First, transfer (by pouring) each bacterial cell suspension to a clean, labeled 50 ml blue-top tube. Pipette in 4 mL of Alkaline Lysis Buffer P2 to each tube, replace the cap (close tightly) and immediately mix by inverting the closed tubes 4-6 times (do not shake). DO NOT VORTEX (this will shear genomic DNA into fragments that will co-purify with plasmid DNA, which we do not want). Do not allow the lysis reaction to proceed for more than 5 minutes, at

room temperature. The aliquot of Buffer P2 should be re-closed immediately after use, to avoid acidification from CO2 in the air. (The cell suspension should turn uniformly blue after addition of Lysis Buffer P2 and appropriate mixing, due to the “LyseBlue” reagent that was added to Buffer P1.)

During this ~5 min incubation in P2, label your prepared QIAfilter Syringe Cartridges (one cartridge for each sample); make sure they are nearby as you perform the next step.

4. Precipitate out the larger cell components: After the ~5 min incubation in P2 is complete, pipette in 4 mL of chilled Neutralization buffer (P3) to each lysate, re- cap tubes, and mix immediately and thoroughly by vigorously inverting the closed tube 4-6 times (don’t shake!!). Immediately after mixing is complete, pour each lysate into the barrel of the appropriately labeled, capped QIAfilter Syringe Cartridge. Leave at room temperature for at least 10 minutes, without disturbing. (Addition of Buffer P3 to the blue solution turns it uniformly colorless after appropriate mixing.) This step neutralizes the suspension and enhances precipitation of detergent, plus bacterial genomic DNA, proteins, and larger cell debris. (You will see a fluffy white material form, and the lysate should become less viscous).

During the 10 minute incubation, set up and label a QIAGEN-tip 100 (containing the silica-DEAE anion exchange resin) for each sample, so that it is suspended over a 50 ml “waste” tube (using the purple holders). Equilibrate (prime) each column by
adding 4 mL of Equilibration buffer (QBT) to each column. Allow it to empty completely into the waste tube by gravity flow. The drained columns will not dry out, and are safe to leave in this manner for awhile if necessary.

5. Bind plasmid DNA to the column: After the 10 min incubation in P3 buffer is complete, remove the little white cap from the QIAfilter Syringe Cartridge outlet nozzle. Position the syringe over a prepared (equilibrated) Qiagen-tip silica column. Gently insert the plunger into the cartridge and filter the cell lysate directly into the equilibrated, drained, and labeled QIAGEN-tip column. Filter until all the lysate has
passed through the cartridge, but do not apply extreme force (i.e., stop if the pressure gets too high; the goal is to keep the white precipitate out of the column).

Allow all of the cleared lysate (containing plasmid) to enter the resin by gravity flow. (i.e., wait until the column has completely drained before proceeding to the wash step, below).

6. Wash out impurities from the column: Wash each QIAGEN-tip twice, each time with 10 mL of Wash buffer QC. Allow the buffer to move through the tip by gravity flow, and wait for the column to drain completely before adding the second wash. All column flow-through solutions to this point can be discarded. (When your waste tube gets too full, simply empty it into the large plastic beaker at your station, and return the tube under the column.)

7. Elute plasmid DNA (remove DNA from column): After the second wash is complete, place a clean, labeled 50 ml tube under each column; elute each DNA sample by adding 5 mL Elution buffer (QF) to each column. After elution, transfer (by pipetting) each DNA solution into a clean, labeled high-speed polypropylene centrifuge tube.

8. Desalt and precipitate plasmid DNA: Precipitate DNA by adding 3.5 mL of room- temperature isopropanol to each eluted DNA sample. Cap the tubes (tightly!) and
mix gently by inverting several times. To ensure that the tubes are perfectly balanced prior to centrifugation, “eyeball” the level of liquid in your tubes, side-by-side, and if necessary, add ~ more isopropanol as needed to bring each tube to the same level (re-mix the tubes if you add more isopropanol). Centrifuge at 15,000 x g for 30 min at 4°C, making sure that your 2 balanced tubes are directly opposite each other in the rotor.

Immediately after the spin, locate your DNA pellet at the bottom edge of the tube. Carefully decant the supernatant without disturbing the pellet, using the provided beaker in the centrifuge room. (Instructor will assist with this step.) NOTE: Isopropanol pellets are typically “small” white pellets and may be difficult to see, in contrast to the fluffy, seemingly denser pellets that result from ethanol precipitation. Isopropanol pellets are also more loosely attached to the side of the tube, and extreme care should be taken when removing the supernatant.

9. “Wash” the pellet of excess salt: With a p1000, add 1000 ul of 70% room- temperature ethanol to each DNA pellet. Disperse the pellets by gentle pipetting and transfer each suspension to a separate, clean, labeled 1.5 ml eppendorf tube.
(These eppendorf tubes are your ‘final’ tubes; please label them legibly with your team name and gene.)

10. Spin 5 min at maximum speed in the microfuge. Note that you are not “dissolving” the DNA pellet at this stage; you should still see “chunks” of precipitate. The 70% ethanol “washes” the DNA pellet by removing (dissolving) the precipitated salt that isopropanol left behind, and it replaces isopropanol with the more volatile ethanol, ultimately making the DNA easier to redissolve.

11. Carefully remove and discard the 70% ethanol with a p1000; remove as much ethanol as possible without disturbing the pellet. If necessary, follow the p1000 with a smaller micropipette (i.e., a p200 or p20) to carefully remove most of the remaining ETOH.
Air-dry the pellet by placing the tube on its side, on a clean paper towel, for 10-15 minutes (no longer).

12. Gently redissolve each DNA pellet in 100 ul (or less) of your TE buffer, pH 8.  Redissolve by thoroughly rinsing the walls of the tube to recover all the DNA.

Composition of buffers in the Qiagen kit:

Buffer P1 (resuspension buffer)
50 mM Tris-Cl, pH 8.0
10 mM EDTA
100 mg/ml RNase A (plus LyseBlue reagent)

Buffer P2 (alkaline lysis buffer)
200 mM NaOH, 1% SDS (anionic detergent)

Buffer P3 (neutralization buffer)
3.0 M potassium acetate, pH 5.5

Buffer QBT (equilibration buffer)
750 mM NaCl
50 mM MOPS, pH 7.0
15% isopropanol
0.15% Triton X-100 (non-ionic detergent)

Buffer QC (wash buffer)
1.0 M NaCl
50 mM MOPS, pH 7.0
15% isopropanol

Buffer QF (elution buffer)
1.25 M NaCl
50 mM Tris-Cl, pH 8.5
15% isopropanol