Because of the instability of the Olson cosmids, we have divised a miniprep procedure that minimizes E. coli growth, thus allowing the least possible time for cosmid DNA deletions/rearrangements to arise and be selected. The amount of cosmid DNA prepared is modest, but is more than enough to make an M13 library. While most genomists focus on the various published biochemical procedures for preparing cosmid DNA, they overlook microbiology, and the microbiology is the most important part of preparing cosmid DNA. If you don't select the "right" E. coli colony, you cannot prepare the "right" cosmid DNA.
1-Late in your working day streak the E. coli (from frozen stocks) on LB + 20 µg/ml carbenicillin (or equivalent) plates. These plates should be reason ably fresh; less than 3 weeks old if stored at 4°C in the dark. Antibiotics decompose with time. For month old plates, the effective antibiotic concen tration may be well below 20 µg/ml. Incubate the plates at 37°C overnight. First thing in your working day , look at your plates. You want small colonies. Usually, these are apparent after overnight incubation. If the colonies are minute, continue to incubate at 37°C, checking hourly. If you see all large colonies, proceed with caution, as you may have E. coli containing a cosmid with deletions. Transfer the plates to room temperature, so that the E. coli does not overgrow.
Late in your work day, inoculate one SMALL colony into 5 ml of LB + 50 µg/ml carbenicillin (freshly made). The antibiotic concentration is increased to main tain strong selection. Do not use day-old colonies/plates nor old medium, they will do you no good. Place the tubes at 37°C, with vigorous aeration, over night.
2-The miniprep procedure is close to that described by Sambrook et. al. (1991, pg. 1.25; Cold Spring Harbor Press), hereafter referred to as the "CSH" proce dure. First thing in the morning, harvest the E. coli and prepare cosmid DNA. The 5 ml culture is divided into three 1.5 ml eppendorf tubes on ice.
These next several steps should be done on ice and with a refrigerated microfuge, if possible. They will work with a microfuge at room temperature, as long as the samples are kept on ice between microfuging and appropriate solutions are pre-cooled. Do not extend microfuge time, as the samples warm during cen trifugation. 1.5 ml of E. coli are pipetted into a 1.5 ml eppendorf tube, which is spun at maximum speed in a microfuge for 30 sec. On the one hand, you want to collect all the E. coli ; on the other hand, packing the bacteria very tightly will make their resuspension difficult. The supernatant is removed by GENTLE aspiration. Careful: do not dislodge the E. coli pellet. Use gravity to supplement aspiration: i. e., start the aspiration with the eppendorf tube ver tical, but invert the tube during aspiration. At the finish, the eppendorf tube is upside down, and gravity is helping to flow the supernatant into the aspira
tor. Keep the working end of the aspirator away from the E. coli pellet.
Note: We use pipets for measuring volume (e.g., 0.1 ml rather than 100 µl). Using a pipet is easier and faster than using a Pipetman, and, if you are careful, pipet accuracy is sufficient.
3- To remove all the traces of the spent bacterial growth medium, add 0.5 ml of STE buffer to each eppendorf tube. Resuspend the bacteria by vortexing vigorously. Spin down the E. coli (microfuge, 30 sec.) and aspirate, as de scribed in step 2. The CSH procedure lists the STE wash as "optional". Our experience says that this step really helps the purification procedure and should be mandatory. In addition, if traces of STE are left with the pellet, it's OK; whereas traces of spent medium are not.
4-Add 0.1 ml of cold solution I. Resuspend the bacteria by vigorous vortexing. The resuspended E. coli must be present as individual cells, and not as clumps, for the next (lysis) step to work efficiently. Keep the eppendorf tubes on ice.
5-Make solution II fresh and at room temperature. Do not use yesterday's solution II, as NaOH will hydrolyze SDS over time. SDS can precipitate at 4 °C, so keep solution II at room temperature. Add 0.2 ml of solution II to each eppendorf tube. Do not vortex. Close the eppendorf tube tightly and invert the tube five times rapidly. The solution should become highly viscous. Keep on ice. The combination of NaOH and SDS lyses the cell and denatures the DNA. Both the cosmid DNA and the E. coli DNAare denatured. However, only the cosmid DNA is supercoiled. Its two strands cannot separate, as they are constrained topologically. When the solution is brought to neutral pH, the cosmid DNA renatures spontaneously, while the E. coli DNA does not renature under these conditions. In addition, most of the E.coli DNA is complexed with membranes. SDS binds strongly to protein (including membrane), and dena tures protein.
6-Neutralize the base with solution III. Add 0.15 ml of cold solution III. This amount of solution III must be mixed thoroughly into the highly viscous lysate, but without shearing the DNA. Therefore, invert the eppendorf tube, and in that inverted position, vortex gently (not vigorously). Place the tube on ice for 5 min.
Now at neutral pH, cosmid DNA renatures. In the presence of SDS and salt, most protein and protein-E. coli DNA complexes precipitate.
Most (if not all) of the E. coli DNA is complexed to cell membrane and SDS and is precipitated by the salt in solution III. To remove these complexes, microfuge at maximum speed for 5 min. Most of the cosmid DNA will remain in the supernatant. Transfer the supernatant (approximately 0.4 ml), to a clean microfuge tube. We use a P200 for the transfer, so as not to disturb the pellet.
Hereafter, we work at room temperature. The completed six steps above is a stopping point, if necessary. Once you start, you must reach this point before stopping or suffer a large reduction in yield.
7-Contaminating protein is still present, despite the SDS precipitation. To remove protein, add an equal volume (usually 0.4 ml) of a phenol: chloroform solution (1:1, with the phenol previously equilibrated with TE). Vortex vigor ously for 10 sec. Microfuge at maximum speed for 5 min. Transfer the aque ous, upper phase (approximately 0.3 ml) to a fresh eppendorf tube. Protein should be within the phenol layer and at the interface. Don't be greedy and try to get every last 50 µl of the upper, aqueous phase. You will also get protein. The CSH procedure lists this extraction step as "optional". However, our expe rience is that without this step, many cosmid DNAs cannot be digested by restriction enzyme.
8-There are traces of phenol in the aqueous phase. We want to get rid of these as soon as possible, because phenol is an oxidizing agent. In addition, we want to purify and concentrate the cosmid DNA by precipitation. The contami nating RNA present will act as a carrier for the DNA precipitation, as the effi ciency of precipitation (recovery) is a (loose) function of nucleic acid mass (DNA and RNA). In addition, alcohol-soluble impurities (e. g., phenol) will be removed. (There is plenty of salt still present from solutions II and III.) Add two volumes (2 x 0.3 ml = 0.6ml) of cold 95-100% ethanol. Vortex 10 sec to mix. Allow the mixture to stand for 2 min on ice. Microfuge at maximum speed for 5 min.
At this point, the CSH procedure calls for removal of the supernatant by "gentle aspiration". We strongly recommend AGAINST using aspiration. Even the gen tlest aspiration can dislodge and suck up the nucleic acid precipitate. We have an easy alternate procedure.
After the microfuge spin, gently pour off the supernatant. A small amount of residual supernatant usually remains and is not a problem. Gently add, so as not to dislodge the precipitate, 1 ml of cold 70% ethanol. That amount of ethanol (70%) will keep the nucleic acid from dissolving , while that amount of water (30%) will dissolve excess salt and other impurities, including the re sidual supernatant from the precipitation. Microfuge at maximum speed for 5 min. Gently pour off the supernatant. Gently add 1 ml of cold 95-100% etha nol. The alcohol will dissolve residual water. Microfuge at highest speed for 5 min. Gently pour off the supernatant. Drain briefly and air dry; there is no residual liquid (buffer or organic ). Dissolve the nucleic acid in 0.2 ml TE. Dissolving may take a few minutes, even at room temperature.
9-The original 5 ml culture was split into three eppendorf tubes. Recombine the DNA from three to one tube (containing 0.6 ml). You have reached a safe stopping point, if necessary. The consolidated nucleic acid in TE can be stored at 4°C for many days. The cosmid DNA must be purified from RNA. Add 30 µl of RNase (4 mg/ml). The RNase must be free of DNase. Fortunately, DNase is very heat labile, and RNase is heat stable. Place your RNase stock solution
at 60 - 65°C for 1 hr. Any contaminating DNase activity will be gone, and approximately 80% of the RNase activity should remain.
Incubate for 1 hr at 37 °C to remove the RNA by digestion. Add 30 µl of proteinase K (600 µg/ml). Incubate for 1 hr at 37°C to digest contaminating protein (including RNase and proteinase K, itself). Cool to room temperature. Add 0.5 ml of phenol: chloroform (1:1). Vortex 10 sec. Microfuge for 5 min. Decant the upper, aqueous phase (approximately 0.6 ml) to a Centricon -30. We use a P200 to effect the transfer. During decanting, don't be greedy. As you near the interface, you may draw denatured protein from the interface.
10-The Centricon is a contemporary form of dialysis. Read the manufacturers' (Amicon) specifications for maximum speed for the particular Centricon and rotor combination that you are using. We use a Centricon -30 and an SS34 rotor in a Sorvall RC-5B centrifuge (at 10°C). Speed for this combination is 5000 RPM. The Centricon -30 holds (approximately) 2.5 ml. ( Note: the Centricon is made of plastic that is resistant to traces of phenol and chloro form, but is easily dissolved by traces of isoamyl alcohol. Many recipes for phenol: chloroform include isoamyl alcohol as an anti-foam reagent. Do not use that recipe.)
Spin the Centricon -30 ( in an SS34 rotor in an RC-5B centrifuge, or equivalent) at 5000 RPM and 10°C. (Other rotor/centrifuge combinations may require a different maximum speed. See the Centricon literature supplied by Amicon Co. for specifications.) 10°C was chosen to avoid "icing-up" the centrifuge, which can occur at 4°C. Under these conditions, it will take approximately 0.5 -1.0 hr to load the sample. We do the three 2.5 ml washes with TE. Each wash takes approximately 0.5-1.0 hr under these conditions. It is very important to remove salt, ribonucleotides (produced by RNase), amino acids (produced by proteinase K), and other impurities for efficient subcloning of the cosmid DNA. After the final TE wash, the Centricon is spun for an additional 2 hr (or more) to achieve minimum volume, approximately 30-40 µl. DNA is recovered as described by Amicon. Of the approximately 35 µl of cosmid DNA, we use 8 µl for double digestion with Eco RI/Hind III to compare our pattern with Olson's pattern.
Solution I: (sterilize by autoclaving or filtration)
Solution II: (must be prepared fresh from stock)
final conc. / stock conc.
Solution III: The final solution is 3 M with respect to potassium and 5 M with respect to acetate.
The glacial acetic acid should be dispensed in a fume hood. Wear safety goggles and gloves.
Phenol: chloroform (1:1; volume: volume): These two compounds are highly toxic. You must wear gloves and goggles. Waste must be disposed of in the fume hood.
First, the phenol must be equilibrated with TE buffer. We are using Amresco phenol, which comes with 2 small bottles of concentrated buffer. Add the bottle of TE buffer to the phenol. (Work in the fume hood!) Shake. Allow the solution to equilibrate overnight in the refrigerator. Phenol is easily oxidized to quinones, which you do not want. Therefore, all phenol solutions are stored in amber bottles and in the refrigerator . To make phenol: chloroform, mix an equal volume of buffer-saturated phenol with chloroform. Work in the fume hood! Shake. Allow the mixture to come to equilibrium by overnight storage in a refrigerator.
Many recipes for phenol: chloroform call for addition of a small amount of isoamyl
alcohol as an anti-foam reagent. However, the Centricon plastic, while resis tant to traces of phenol and chloroform, is dissolved by isoamyl alcohol. Do not add isoamyl alcohol. Do not use Amresco pre-mixed phenol: chloroform, as it contains isoamyl alcohol.
Ethanol: 70% and 95-100%, stored at -20°C.
Ribonuclease A (RNase): Commercial RNase, even highly purified, can con tain traces of deoxribonuclease (DNase). If DNase were present, it would be disaster for this procedure. Fortunately, RNase is very resistant to heat, and DNase is very sensitive to heat. Therefore, we dissolve the RNase (4 mg/ml) in a buffer at pH 4.5 (where RNase is most stable): 0.1 M sodium acetate (to pH 4.5 with glacial acetic acid - wear gloves and goggles and work in the fume hood!). Heat the RNase solution at 60°C for 1 hr. Aliquot and freeze at -20°C. Alternatively, buy the relatively expensive RNase (DNase-free) from Boehringer /Mannheim.
Proteinase K: Purchase as a highly purified enzyme. Dissolve at 600 µg/ml in TE buffer. Aliquot and store at -20°C.