Working with DNA: Good microbiological practice

Good microbiological technique will always ensure the best yield and quality of plasmid DNA. To prepare the perfect bacterial culture for your plasmid prep, follow the steps below.

1. Prepare a starter culture by inoculating a single colony from a freshly streaked selective plate into 2–10 ml LB (Luria-Bertani) medium containing the appropriate antibiotic. Grow at 37°C for ~8 hours (logarithmic growth phase, see figure Growth of E. coli cultures) with vigorous shaking (~300 rpm).
   
   Tip: Do not inoculate directly from glycerol stocks, agar stabs, or plates that have been stored for a long time, as this may lead to loss or mutation of the plasmid.
   
   Tip: It is often convenient to grow the starter culture during the day so that the larger culture can be grown overnight for harvesting the following morning.
   
   
2. Dilute the starter culture 1/500 to 1/1000 into a larger volume of selective LB medium, as indicated in the appropriate plasmid purification protocol.
   Use a flask of at least 4 times the volume of culture to ensure sufficient aeration.
   Do not use a larger culture volume than recommended in the protocol, as this will result in inefficient lysis and reduce the quality of the preparation.
3. Grow the culture at 37°C with vigorous shaking (~300 rpm) for 12–16 hours (see next section).
4. Harvest the bacterial culture 12–16 hours after inoculation. This corresponds to the transition from logarithmic into stationary growth phase (see figure Growth curve of E. coli in LB medium), when cell density is high (3–4 x 109 cells per ml) and RNA content of cells is low. Harvesting too early may result in lower than expected yields of plasmid DNA due to a lower cell density. Harvesting too late may result in low plasmid quality and yield due to DNA degradation from over-aging of the culture.
   
   Tip: Growth of cultures is dependent on factors such as host strain, plasmid insert and copy number, and culture medium. To determine the optimal harvesting time for a particular system, monitor the cell density and the growth of the culture by measuring the OD600 (see next section).
   
   
5. Harvest the bacterial culture by centrifugation at 6000 x g for 15 min at 4°C. Remove all traces of supernatant by inverting the open centrifuge tube until all of the medium has been drained. The cells are now ready for the lysis procedure, as indicated in the appropriate plasmid purification protocol.
   The procedure may be stopped at this point and continued later by freezing the cell pellets obtained by centrifugation. The frozen cell pellets may be stored at –20°C for several weeks.

The growth curve of an E. coli culture can be divided into several distinct phases. The first, lag phase, occurs directly after dilution of the starter culture into fresh medium. During this phase, cell division is slow as the bacteria adapt to the fresh medium. The bacteria then start to divide more rapidly and the culture enters logarithmic (log) phase (4–5 hours after dilution), during which the number of cells increases exponentially. As the available nutrients in the medium are used up and released metabolites inhibit bacterial growth, the culture becomes saturated and enters stationary phase (~16 hours after dilution), during which cell density remains constant. Eventually the culture enters the phase of decline as cells start to lyse, the number of viable bacteria falls, and DNA becomes partly degraded.

There are different methods for storing E. coli strains depending on the desired storage time. Glycerol stocks and stab cultures enable long-term storage of bacteria, while agar plates can be used for short-term storage. Preparation instructions and useful tips for each of these methods are given below.

Glycerol stocks

E. coli strains can be stored for many years at –70°C in 15% glycerol.

Prepare glycerol stocks of bacteria as follows:

1. Add 0.15 ml glycerol (100%) to a 2 ml screw-cap vial and sterilize by autoclaving.
   
   Tip: Vials of sterilized glycerol can be prepared in batches and stored at room temperature until required.
   
   
2. Add 0.85 ml of a logarithmic-phase E. coli culture to the vial of pre-sterilized glycerol.
3. Vortex the vial vigorously to ensure even mixing of the bacterial culture and the glycerol.
4. Freeze in ethanol–dry ice or liquid nitrogen and store at –70°C.
   
   Tip: Avoid repeated thawing and re-freezing of glycerol stocks as this can reduce the viability of the bacteria.
   
   Tip: For precious strains, storage of 2 stock vials is recommended.
   
   Tip: When recovering a stored strain, it is advisable to check the antibiotic markers by streaking the strain onto a selective plate.

Stab cultures

E. coli strains can also be stored for up to 1 year as stabs in soft agar. Stab cultures are used to transport or send bacterial strains to other labs.

Prepare stab cultures as follows:

1. Prepare and autoclave LB agar (standard LB medium containing 0.7% agar).
2. Cool the LB agar to below 50°C (when you can hold it comfortably) and add the appropriate antibiotic(s). While the agar is still liquid, add 1 ml agar to a 2 ml screw-cap vial under sterile conditions, then leave to solidify.
3. Vials of agar can be prepared in batches and stored at room temperature until required.
4. Using a sterile straight wire, pick a single colony from a freshly streaked plate and stab it deep down into the soft agar several times (see figure Inoculating a stab culture).
5. Incubate the vial at 37°C for 8–12 h leaving the cap slightly loose.
6. Seal the vial tightly and store in the dark, preferably at 4°C.
7. When recovering a stored strain, it is advisable to check the antibiotic markers by streaking the strain onto a selective plate.

Agar plates

Plates of streaked bacteria can be sealed with Parafilm and stored upside-down at 4°C for several weeks. Bacteria should always be streaked onto plates containing the appropriate antibiotic to ensure that selective markers are not lost.

To obtain well-isolated colonies, streak an agar plate as follows:

1. Flame a wire loop, and cool on a spare sterile agar plate.
2. Using the wire loop, streak an inoculum of bacteria (from a glycerol stock, stab culture, or single colony on another plate) across one corner of a fresh agar plate, as shown in the figure Streaking bacteria on agar plates.
3. Flame and cool the wire loop again. Pass it through the first streak and then streak again across a fresh corner of the plate.
4. Repeat again to form a pattern.
5. Incubate the plate upside down at 37°C for 12–24 hours until colonies develop.