Preparation of Liquid Media

LB medium

This is a standard liquid medium used to grow cultures of E. coli. The recipe can be found in Molecular Biology Reagents. All solid reagents should be added to the appropriate size Wheaton bottle (or other autoclavable container), and water added. When most of the solids have been dissolved (it is not necessary to completely dissolve all the solids) the solution should be neutralized by the addition of the indicated amount of NaOH. The bottle should be loosely capped—which should be secured from untwisting with tape—and autoclaved for 20 minutes at 250 °F. The solution should cool to 50 °C before any antibiotics, if needed, are added. (If you are able to hold the bottle in an ungloved hand without discomfort, it is cool enough for the addition of antibiotics.) After the addition of antibiotics, the solution should be swirled vigorously to mix thoroughly.

Media prepared without antibiotics can be stored at room temperature until opened and used the first time. Thereafter media should be stored in the refrigerator. Media prepared with antibiotics, especially ampicillin, should be stored in the refrigerator immediately.

TB medium

This is a standard “rich” medium used to grow E. coli cultures to very high cell density, and is thus useful for protein overexpression. The recipe can be found in Molecular Biology Reagents. It is important to note that the nutrient media and the phosphate buffer solutions must be prepared and autoclaved separately. Once the solutions have cooled, they are combined using sterile technique, and antibiotics and any other additives added at that time. This medium should be swirled vigorously to mix and stored in the refrigerator if not used immediately.

Preparation of agar plates

LB agar plates

Preparation is exactly as for LB medium, except that 15 g of agar is added to the mixture prior to autoclaving. After removal from the autoclave, the solution should be swirled to ensure even mixing of the agar, which will be concentrated at the bottom of the bottle. The solution must be cooled to 50 °C before adding antibiotics. Swirl to mix, but avoid introducing bubbles. Pour about 20 mL of the medium into 90 mm plates—just enough to cover the entire bottom of the plate about 2-3 mm deep—working quickly so that the agar medium does not harden in the bottle. Flame the neck of the bottle occasionally to maintain sterility. One liter of LB-agar should be sufficient to pour 40-50 plates. Plates should be appropriately marked to identify the antibiotic, if any, used in the medium. A suggested code is one black stripe on the edge of the lid to indicate an LB plate, two red stripes to indicate an LB-ampicillin plate, three green stripes to indicate an LB-chloramphenicol plate, etc.) Allow the plates to harden. If the plates “sweat” excessively, they may be placed in a 37 °C incubator overnight to remove condensation. Plates should be stored inverted at 4 °C, and warmed to room temperature prior to use.

Streaking out cultures from frozen glycerol stocks

Remove an LB plate with the appropriate antibiotic, if necessary, and warm it to room temperature. If necessary, remove condensation by incubation in a 37 °C incubator. Label the bottom of the plate with your initials, date, and strain of E. coli used. For example the label

RSR

3-17-99

pARCANX/JM109

indicates that individual RSR streaked out a culture of E. coli strain JM109 harboring the pARCANX plasmid on March 17, 1999. If this plate is discovered in the incubator or refrigerator in June 1999 someone will realize that it needs to be discarded!

Sterilize an inoculating loop by heating in a flame until it glows cherry red over its entire length. Allow it to cool before using it to streak samples. Remove the cryovial containing the frozen glycerol stock from the –80 °C freezer, and without letting the sample thaw, take the sterile inoculating loop and scrape a little bit of ice from the surface of the frozen stock. You do not need much material: if you can see it on the loop, you have too much. Take the inoculating loop and touch it to the agar plate near one edge. In one continuous motion, drag the loop across the plate 20-25 times, as shown in Figure 1:

Figure 1. Streaking an agar plate to obtain single colonies (clones).

Replace the lid, invert the plate, and incubate overnight at 37 °C. If the plate has been streaked correctly, the bacterial growth will be nearly continuous and confluent near the beginning of the streak, but will thin out into single colonies somewhere farther down the plate. These single colonies represent growth from a single bacterial cell.

Streaked plates may be stored for a several weeks in the refrigerator. To keep plates from drying out, cut a strip of parafilm 1 x 10 cm and stretch it tightly around the edge of the plate, sealing the lid to the bottom. For longer term storage of bacterial cultures, frozen glycerol stocks should be prepared. Store plates inverted to prevent condensation from dripping on the agar surface.

Preparation of frozen glycerol stocks of E. coli

An overnight culture of E. coli should be shaken by hand to resuspend cells thoroughly. Using sterile technique, pipet 0.5 mL of bacterial culture into a sterile 1.5 mL cryovial, and dilute with an equal volume of sterile 30% glycerol. Cap the vial tightly and mix the contents of the vial completely by inverting the vial repeatedly. Glycerol stocks may be stored for a day or two at –20 °C but should normally be immediately frozen at –80 °C. Frozen stocks at this temperature, and not subjected to repeated thawing and freezing, will remain viable nearly indefinitely.

Preparation of competent cells

Standard method

The competent cells produced according to the following procedure¹ are good for routine transformations, and typically yield approximatey 10⁶-10⁷ transformants per ug of DNA. High efficiency competent cells (>10⁸ transformants per ug DNA) are generally required for transformations of ligation mixtures, and are best purchased commercially.

• Streak out E. coli JM109 (or other strain) on an LB plate. Incubate overnight at 37 °C.
• Transfer a colony into 5 mL of LB medium in a 50 mL conical tube. Shake overnight at 37 °C.
• Inoculate 5 mL of LB medium with 50 uL of overnight culture and incubate at 37 °C with shaking to an OD600 of 0.3-0.4, about 2 hr. (The exact optical density is not that critical—even overnight cultures will produce reasonably competent cells.)
• Add an equal volume of ice-cold sterile 2x TSS buffer (20% PEG 8000-40 mM MgSO4-10% DMSO) and incubate for 5-15 minutes.
• Quickly dispense 100 uL aliquots of the suspension into chilled, sterile, microcentrifuge tubes. Immediately snap-freeze cells by immersion of the tubes into liquid nitrogen.
• Store tubes at –80 °C until needed.

Notes

1. Chung, C. T., & Miller, R. H. Meth. Enzymol. 1993, 218, [43]

Mix-N-Go competent cells (Zymo Research kit)

These competent cells are suitable for transforming cells already containing a plasmid (e.g. containing chaperone protein genes) with a second plasmid with a different origin of replication and antibiotic resistance selection marker.

• Streak out a cell strain (e.g. JM109) already containing a desired plasmid on a suitable antibiotic plate for proper selection. Incubate overnight at 37 °C.
• Transfer a colony into 5-10 mL of LB medium in a 50 mL conical tube with the appropriate antibiotic for proper selection. Shake overnight at 37 °C.
• Inoculate 50 mL of SOB medium with 0.5 mL of overnight culture, including the appropriate antibiotic for proper selection. Grow cells at 25 °C to an A(600) of 0.4-0.6, about 5-7 hours.
• Pellet cells by centrifugation in a 50 mL conical tube at 2000 xg for 10 minutes
• Resuspend cells gently in 5 mL ice-cold, freshly prepared 1X wash buffer (from kit)
• Pellet cells by centrifugation in a 50 mL conical tube at 2000 xg for 10 minutes
• Resuspend cells gently in 5 mL ice-cold, freshly prepared 1X competent buffer (from kit)
• Aliquot on ice 0.1-0.2 mL of cell suspension in sterile cryovials. Use immediately or store at -80 °C for future use.

Transformation of competent cells

The following transformation protocol works well with home-made and many commercially obtained competent cells.

• Gently warm 50-100 uL of competent cells by hand until just thawed, then place in an ice bath.
• Add 1-10 uL (typically 5 uL) of ligation mixture, or 10-100 ng of DNA. Flick tube gently to mix.
• Place mixture on ice for 15-60 min. (Longer times will generate more transformants.)
• Add 500 uL of LB medium (with no antibiotic) supplemented with 5 uL of sterile 2.0 M glucose, mix gently, and incubate for 1 hr at 37 °C.
• Using a bent glass or stainless steel rod—sterilized by immersion in 95% ethanol flaming off three times— spread an aliquot of 100-400 uL (typically 400 uL) of the mixture on LB plates containing 50 ug/mL ampicillin or other appropriate antibiotic. (Note: if blue-white screening¹ is desired, add 35 uL 2% X-gal and 15 uL 100 mM IPTG to the mixture before spreading.)
• Incubate plates overnight at 37 °C, but not more than 18 hr to prevent satellite colony formation.

Notes

1. Many plasmid vectors, such as pUC18, contain a short segment of E. coli DNA which codes for the first 146 amino acids of the beta-galactosidase gene, lacZ. In the middle of these gene there is a cloning site which inactivates this gene fragment if foreign DNA is inserted there. If such a plasmid is used in a host strain of E. coli, such as JM109, that contains a chromosomal copy of a gene which codes for the complementary C-terminus of beta-galactosidase, the presence or absence of a DNA insert in a clone or bacterial colony can be evaluated by screening for beta-galactosidase activity. If there is no gene insert in the vector of a clone or colony, the intact N-terminus of beta-galactosidase can complement with the chromosomal C-terminus of the enzyme, yielding active protein. On the other hand, if a DNA insert fouls up the plasmid-encoded N-terminus of beta-galactosidase, then the clone or colony will have no beta-galactosidase activity. Clones (colonies) can be screened right on the agar plate if a chromogenic beta-galactosidase substrate and inducing agent is included on the agar plate. X-gal (5-bromo-4-chloro-3-indolyl-beta-D-galactoside) is a colorless compound which turns blue when cleaved by the beta-galactosidase. Thus, clones (colonies) of bacteria which have no DNA insert in the plasmid will appear blue, and those which have DNA insert will appear white. The blue color can be enhanced by leaving the plates out at room temperature for several hours after colonies have formed. Our laboratory rarely uses blue-white screening because the proportion of transformants with DNA insert should be in excess of 90% if the appropriate precautions are taken.

Transformation of Z-competent cells

Z-competent cells (Zymo research) are high efficiency (>10⁸ cfu/ug DNA) commercial competent cells with an extremely simple transformation procedure, described below:

• Pre-warm an agar plate with the appropriate antibiotic selection factor to 37 ºC. This step is critical to efficient transformation.
• Take a single tube (100 uL) of Z-competent cells and thaw on ice.
• Add 1-5 uL of DNA to cells and mix gently.
• Incubate cells on ice for 10-60 minutes. (Longer times will generate more transformants.)
  • If using Zymo Mix and Go cells, transformation is complete in less than a minute.
• If using Zymo Mix and Go cells with an antibiotic other than ampicillin follow these additional steps
  • Add 4 volumes of SOB buffer (400 μL to 100 μL cells) and 1/100 volume (5 μL in 500 μL) 2 M glucose to competent cell mixture
  • Incubate at 37 ºC for one hour with occasional mixing to allow for antibiotic resistance to develop
• Using a bent glass or stainless steel rod—sterilized by immersion in 95% ethanol and flaming off three times—spread an aliquot of 10-100 uL (typically two plates at 10 and 100 uL) of the mixture on LB plates containing 50 ug/mL ampicillin or other appropriate antibiotic.
• Incubate plates overnight at 37 °C, but not more than 18 hr to prevent satellite colony formation.

Transformation of Competent One Shot BL21(DE3) E. coli cells

• Thaw one vial of One Shot® cells on ice per transformation.
• Add 0.5 µL of plasmid DNA to E. Coli cells and mix by tapping gently.
• Incubate the vial(s) on ice for 30 minutes.
• Heat shock the cells by incubating the vial(s) for exactly 30 seconds in the 42°C water bath. Do not mix or shake.
• Remove the vial(s) from the 42°C bath and quickly place on ice.
• Add 250 μL of pre-warmed LB medium and 2.5 µL of sterile 2.0 M glucose to the vial(s). (LB is a rich medium; use proper sterile technique to avoid contamination.)
• Place vials in 37°C warming block for 60 minutes and invert every ~10 minutes.
• Plate two different volumes of the transformation reaction onto LB plates containing the appropriate antibiotic for plasmid selection. Use two volumes: 10 µL & 90 µL to ensure well-spaced colonies on at least one plate. The remaining transformation reaction may be stored at 4°C and plated out the next day, if needed.
• Invert the plates and incubate at 37°C overnight.
• Select transformants from the plates and culture as described on page 8.
  • Clones may exhibit differences in expression of heterologous genes. We recommend choosing 3–4 transformants when characterizing clones for protein expression.

Transformation of ArcticExpress (DE3) Competent Cells

• Thaw the vial of competent cells on ice.
• Preheat 0.9mL LB media with 9.0uL 2.0M glucose at 42°C.
• Dilute XL10-Gold β-mercaptoethanol 1:10 with dH2O. Each aliquot needs 2.0uL of diluted β-mercaptoethanol.
• Swirl and incubate on ice for 10 minutes, swirling every 2 minutes.
• Add 0.5uL of expression plasmid DNA and swirl.
• Swirl and incubate on ice for 30 minutes.
• Heat-pulse the transformation at 42°C for 20 seconds.
• Incubate the reaction on ice for 2 minutes.
• Add 0.9mL of preheated LB medium to each transformation reaction and incubate the reactions at 37°C for 1 hour with shaking at 225-250 rpm.
• Using a sterile spreader, spread 10uL and 90uL on an LB agar plate with ampicillin and incubate at 37°C.