Competent cells, calcium chloride method, E. coli, description

CaCl2 competent cells

Contributor: : Zappe, H

Reference: Dagert and Ehrlich (1979) Gene 6:23-28.

1. Grow 5 ml E. coli in 2 X YT

2. Dilute 1ml culture in 100 ml fresh 2 X YT

3. Grow to OD600 = 0.2 - 0.4

4. Centrifugation at 5000 rpm for 5 min at 4 C

5. Resuspend the cells in 50ml of 0.1 M ice-cold MgCl2.

6. Hold on ice for 30 min

7. Centrifugation at 5000 rpm for 5 min at 4 C

8. Resuspend in 10ml of ice cold 0.1 M CaCl2.

9. Mix and leave on ice for 30 min, then store/use

Transformation

1. To transform, mix 0.1 ml aliquots of cells with DNA (1 - 10 ng). Leave on ice for 10 - 30 min.

2. Heat shock at 42 C for 2 min (90 - 120 s is OK)

3. Add 0.9 ml 2 X YT or LB and allow expression for 30 - 60 min before plating.

4. If using X-gal and IPTG (optional), place 0.1 ml expression mix on the plate, add 40 æl X- gal and 4 æl IPTG, and spread together. This ensures that wherever cells go, X-gal does too!. Better still, add IPTG and X-gal to the plate (0.5 ml X-gal and 50 ml IPTG per 100 ml agar).

DMSO competent cells

Contributor: : Zappe, H

Reference: Chung and Miller (1988) N.A.R. 16:3580.

1. Grow 5 ml overnight of the strain in 2 X YT in a standard container. Screw cap tight and lie flat on the shaker for good aeration.

2. Dilute overnight culture 1/100 - 1/200 to 25, 50 or 100 ml 2 X YT broth in flasks 5 - 10 X culture volume (i.e. 25 ml in 250 ml flask etc.).

3. Grow to early log phase (OD600 = 0.2 - 0.4) (90 - 180 min depending on the strain)

4. Collect cells by centrifugation (4000-5000 rpm for 5 min or 5000 x g for 5 min) at 4 C.

5. Cells are resuspended in 1/10 th culture volume of ice cold TSB and held on ice for 10 min before transformation.

Transformation

1. For transformation, 0.1 ml cells are mixed with up to 0.1 æg DNA and held on ice for 10 min.

2. Add 0.9 ml TSBG and incubate 37 C for 30 - 60 min before plating.

3. Competent cells can be frozen in dry-ice/ethanol and stored at -70 C.

DMSO competent cells

Contributor: : Zappe, H

Reference: Chung and Miller (1988) N.A.R. 16:3580.

1. 5 ml overnight E. coli in 2 X YT

2. Dilute overnight culture 1/100 - 1/200 to 25, 50 or 100 ml 2 X YT broth in flasks 5 - 10 X culture volume (i.e. 25 ml in 250 ml flask etc.).

3. Grow to early log phase (OD600 = 0.2 - 0.4) (90 - 180 min depending on the strain)

4. Collect cells by centrifugation (4000-5000 rpm for 5 min or 5000 x g for 5 min) at 4 C.

5. Cells are resuspended in 1/10 th culture volume of ice cold TSB and held on ice for 10 min before transformation.

Competent cells can be frozen in dry-ice/ethanol and stored at -70 C.

Transformation

1. For transformation, 0.1 ml cells are mixed with up to 0.1 æg DNA and held on ice for 10 min.

2. Add 0.9 ml TSBG and incubate 37 C for 30 - 60 min before plating.

DNA isolation from bacterial cells

Description:

A. The method of Zappe is quite wordy and applies to most bacteria

protocol

B. Large scale bacterial genomic DNA prep by Coyne

protocol

DNA isolation from bacterial cells

Contributor: : Zappe, H

Reference: Based on the method of Marmur (J. Mol. Biol. 3:208-218, 1961). Modified by J.L. Johnson (Virginia Tech.)

The volumes of buffers given are for cultures of 300-600 ml of E.coli type cultures (good growers). Reduce volumes for low density or smaller cultures. Note that mycobacteria may have a similar culture density, but DNA yield would be much lower due to the cell envelope.

1. Harvest the cells by centrifugation at 7500 RPM for 10 min. Decant the supernatant and allow the centrifugation bottles to drain upside down on paper towel. Do this under appropriate conditions for pathogens. Resuspend the cells in 20 ml of suspension buffer for enzymatic lysis or 10 - 20 ml for disruption by French pressure cell treatment. See 2cii for glass bead method. The last two methods require a thin paste of cells - not too dilute. Resuspend the cells properly, either by vortexing the pellet with 1 or 2 ml buffer before adding the rest of the buffer, or by drawing up and expelling the cell/suspension buffer mixture with a 10 ml pipette. There should be no clumps of cells after this step. Transfer the cells to a suitable flask (125 - 150 ml).

2. Digest the cells with lytic enzyme.

2a. Gram-negative bacteria . Add dry lysozyme (4 mg/ml or about 1/8 teaspoon for 25 ml!) and incubate. The time and temperature can vary from 10 min at room temperature to 4 h at 37 C depending on the organism. Remove small samples (100ml) periodically and mix with 100ml lysing solution. If the cells lyse, go to step 3, if not continue incubation. More lysozyme can also be added.

2b. Gram-positive bacteria . Add dry lysozyme (8 mg/ml or about 1/4 teaspoon per 25 ml!) and incubate at 37 C. Start doing the lysis test from 15 min onwards. Other enzymes are also available if lysozyme does not work, but lysozyme is the cheapest.

2c. Recalcitrant bacteria . Some organisms will not be made sensitive to detergent lysis using lytic enzymes and must be physically disrupted. Either of the following methods can be used.

i. French Pressure cell. Pass the cell suspension through the French Pressure cell at 16 000 psi (upper limit for many cells) into an equal volume of lysing solution.

ii. Glass beads . Using a Braun homogeniser with liquid CO2 cooling. Scrape the cell pellet form the centrifugation bottle and place it into a tared Braun shaker bottle. Make up the weight to 20 g with suspension buffer (cells + buffer), add 20 ml glass beads (0.1 mm diameter) and shake for 5 min at 4000 cycles per minute (use the cooling system). Separate the lysate from the glass beads by filtration through a coarse 60 ml scintered glass filter. Use 20 ml lysing solution to wash the last of the lysate from the beads.

1. Add an equal volume of the 2X lysing solution (unless its already been added in step 2c i or ii above) and a volume of 5 M NaClO4 equal to 1/4 of the combined total. This mixture forms a precipitate - heat to 50 C to get it back into solution. If the cells were susceptible to lysis, mix with a swirling action to get a uniform mixture before all the cells lyse. Lysis is evident by the turbid solution becoming translucent and very viscous. Incubate the lysate for 4 h at 50 - 60 C to degrade cellular proteins. As little as 1 h may be OK, but overnight is sometimes convenient and better.

2. Add 15 ml phenol-chloroform solution, shake by hand to form a uniform emulsion and then shake on a wrist action shaker for 20 min. Transfer the mixture to a 50 ml centrifuge tube and centrifuge at 12 000 RPM for 10 min at room temperature. Meanwhile rinse the flask and let it drain. Slowly decant as much of the upper aqueous layer back into the flask as you can. Do not allow any of the organic phase to go over. The remainder of the aqueous phase can be collected by using an inverted 2 or 5 ml glass pipette and a pi-pump. Do not use narrow bore pipettes or Gilson tips as significant shearing of the DNA can occur. Repeat the extraction procedure twice more. Extraction of proteins is complete if there is little or no white precipitate at the interface after the centrifugation step.

3. Carefully decant the aqueous phase from the last phenol-chloroform extraction into a suitable flask (125 ml Erlenmeyer). Taring the flask and then weighing the solution is a good way of estimating the volume (1 ml = 1 g). Add 0.6 volume of Isopropanol and swirl to mix. The nucleic (DNA and RNA) will precipitate and form a loose clot (unless the cells were physically disrupted, where it will be necessary to centrifuge the solution). Hold the clot back with a Pasteur pipette and pour off the lysate-Isopropanol solution. Add 25 ml 76 % ethanol and allow to stand for 10 min. Decant the ethanol and repeat the wash. Decant the ethanol and press the clot of nucleic acid with a Pasteur pipette to get rid of most of the ethanol. Allow the precipitate to dry at 37 C for 15 min. Never centrifuge preparations of high Mol. wt. DNA - they take a very long time to resuspend (days).

4. Dissolve the nucleic acid precipitate in 20 ml TE buffer (may take a few hours), add 0.25 ml of RNase mix and incubate at 37 C for 1 h (Note that in any nucleic acid preparation, most of the nucleic acid at this point is RNA). Extract the solution once with 5 ml chloroform:isoamyl solution, centrifuge and save the aqueous layer.

5. Add 0.1 volumes of 3 M Sodium acetate, mix, overlay with 2 volumes of 95% ethanol, and collect the DNA by spooling onto a glass rod (or Pasteur pipette) by rotating the rod and stirring the solution at the same time. Continue until both phases are completely mixed. Alternatively, the DNA can be precipitated just by swirling the solution as in 5 above. Collect the "clot" of DNA, wash and dry the precipitate as in 5 above.

6. Dissolve the DNA in 3 - 5 ml TE (again, this could take a day - leave at 4 C). Do a 310 - 220 nm scan to determine the actual concentration. Store at a concentration of at least 0.5 mg/ml at -20 C.

Note: The procedure can be stopped and the DNA solution stored at 4 C for up to a few days anyhwere after step 4. However, do not store solutions containing phenol - decant the aqueous phase and store this.