PreparingLambdaDNA

Preparing Lambda DNA

1- Coliphage lambda DNA is a widely used vector for recombinant DNA. The middle third of its 48,000 bp contains no genes required for lytic growth and is, therefore, replaceable. The usual recombinant lambda DNA contains 80% lambda vector DNA and 20% insert, as compared to a usual cosmid DNA that contains 10% vector and 90% insert.

Wild-type lambda is not very lytic, compared to coliphages T4 or T7. Recombinant lambda phage are usually less lytic than wild-type. We recommend growing recombinant lambda phage on large (150-mm diameter) plates, rather than in liquid culture. Beginners, in particular, may not be aware sufficiently that in liquid culture E. coli can easily overgrow phage lambda, rather than the desired vice versa.

2- Traditionally, the E. coli host for lambda is grown on NZY medium. This medium is not as rich as LB (and many other) medium, and E. coli growth is slower, making it even less likely that E. coli will overgrow the phage.

The lambda receptor on the bacterial cell outer surface is part of the maltose-usage pathway. To induce the receptor to high levels (10²-10³ receptors per cell), maltose is added to a final concentration of 0.2% in the medium.

When growing lambda on plates to prepare DNA (as opposed to picking plaques or titering), agarose is substituted for agar. Agarose is much more expensive than agar, but does not contain impurities that inhibit enzymes. Lambda DNA prepared from agar plates may not be digestible by restriction enzymes because of the presence of inhibitors that have leeched from the agar.

3- The host for lambda is E. coli C600 and its subsequent variants. To avoid complications, the E. coli C600 strain is usually restriction-negative: r-m- or r-m+.

For our purposes, we find it useful to start with a fresh E. coli colony (on NZY or LB plates). The colony is picked into 5 ml of NZY medium plus 0.2% maltose and placed on a wheel at 37°C for approximately 4 hr. (This time will be much longer if the colony is from an old plate or has been stored at 4°C or frozen.) Using sterile conditions, the culture is transferred to a sterile 15-ml conical tube. The E. coli are pelleted by centrifugation in an SS34 rotor with adaptors in an RC-5B centrifuge (or equivalent) for 8 min at 4 K rpm (or equivalent). The medium is poured off gently. The bacterial pellet is resuspended in 4-6 ml of 0.01 M MgSO4 by pipetting up and down (using sterile conditions), NOT by vortexing. The concentration of bacteria is adjusted to yield an "optical density" (really, light scattering) of 2.0 at wavelength 600 nm. E. coli prepared in this way can be stored at room tempera ture and used for up to one week. To resuspend the E. coli, gently pipet the cells. DO NOT VORTEX. Cells prepared in this manner are employed at values of 10 µl per small or 20 µl per large plate.

4- At times, we shall refer to the procedures published in Sambrook, Fritsch, and Maniatis, in "Molecular Cloning: a laboratory manual", pp. 2.60-2.79, second edi tion, 1991, as the "CSH" procedure or the "CSH" book.

One common source of confusion when comparing methods for growing lambda concerns "SM buffer" and whether or not it contains gelatin. In our protocol, "SM buffer" refers solely to Tris buffer with magnesium salt and NaCl. If gelatin is to be added, we state so explicitly in the few cases where we use it.

5- The start of any procedure for preparing lambda DNA is to pick a single, well -isolated plaque. (A lambda plaque contains phage and lysed bacteria and appears relatively "clear" against the bacterial lawn.) For the Olson frozen lambda stocks, we use sterile technique to scrape a small amount (approximately a toothpick-full) of frozen stock into 1 ml of SM buffer plus gelatin. (The gelatin helps stabilize the phage.) From this one ml, we make 10-fold dilutions (in SM buffer plus gelatin) through 10-4. In general, we plate 1, 10, and 100 µl of the 10 -3 and 10-4 dilutions.

There is significant preparation time. NZY agar plates should be placed at 37°C, so that they are warm when used. NZY top agar (3 ml per plate) should be melted and placed at 50°C (just warm enough to keep it liquid, and not so hot as to kill the E. coli). E. coli (10 µl) and 0.2 ml SM buffer (no gelatin) should be mixed in a small tube, one tube per agar plate.

Phage are added to the E. coli and SM buffer, and the tubes are placed on the 37°C wheel for 20-30 min. The kinetics of lambda attachment to its receptor are fairly slow; the 20 min incubation allows time for this reaction to occur. Add the 3 ml of top agar to the mixture of phage and bacteria. Vortex for 10 sec. Pour onto an agar plate. Rock the plate by hand to reach an even distribution of top agar. Allow the top agar to solidify at room temperature (approximately 10 min). Place the inverted plates at 37°C overnight. The next day, at least one plate should have well-isolated plaques. If there are too many plaques, start over with more dilution. If there are too few plaques, start over with less dilution.

For picking plaques, we find the following procedure simple and straightforward. Autoclave inverted Pasteur pipets (without cotton plugs). Holding the narrow end, punch out a plaque with the (relatively) wide-mouth end. The agar stays in the pipet and can be expelled into a sterile eppendorf tube by a wrist flick. Add 0.5 ml of SM buffer (no gelatin) and 2 drops of chloroform (the chloroform stops the E. coli from growing). To elute the phage, allow the eppendorf tube to stand for 2-4 hr at room temperature or at 4°C overnight.

6- A phage stock is made from the isolated plaque. Again, NZY agar plates are at 37°C; NZY top agar is at 50°C; 0.2 ml of SM buffer plus 10µl of E. coli are in small tubes. Add 5-20 µl (or more) of phage, depending on plaque size, to the E. coli and SM buffer. Place on a 37°C wheel for 20-30 min. Add NZY top agar and vor tex for 10 sec. Pour onto a warm NZY agar plate. After the top agar hardens (approximately 10 min), invert the plate and place at 37°C overnight.

The next morning the plates should show a fully infected, disrupted E. coli lawn. If you see individual plaques, the inoculum was too low. Toss the plates, and repeat the infection with a larger volume of the plaque. If the infection looks confluent, add 5 ml of sterile SM buffer (no gelatin). Make sure that the plate is completely covered with buffer. Gently rotate or rock the plates at room temperature for 1-2 hr. Harvest the buffer (using a sterile pipet) into a sterile conical 15-ml tube. The "milky" appearance is caused by the presence of bacterial debris. Add 0.2 ml of chloroform and vortex for 10 sec. Spin the tube in an SS34 rotor with adaptors, RC-5B centrifuge (or equivalent), at 4 K rpm for 8 min. Decant the clear yellow supernatant into a fresh, sterile, 15-ml conical tube. Add a drop of chloroform (the chloroform is to stop the growth of residual E. coli) and store at 4°C. These stocks are stable for several months at 4°C. Frozen (-70°C) stocks can be made by mixing 0.1 ml DMSO with 1.4 ml of lambda phage stock.

7- The phage stocks of Olson lambda are usually in the range 10 5-106 pfu (plaque-forming units) per µl. These numbers are lower than those given in CSH, pg. 2.65; the Olson lambda phage grow very poorly. Our stocks have been as low as 2x10 3 and as high as 8x107 pfu per µl. The general correlation is that phage that pro duce small plaques tend to yield stocks below 105 pfu per µl; phage that produce (relatively) large plaques tend to yield stocks over 10 6 pfu per µl; but the correlation is not absolute.

The lambda stocks are diluted ten-fold (as appropriate: 10 -2, 10-3, etc.) in SM buffer plus gelatin. NZY agar plates are at 37°C. NZY top agar is melted, aliquoted, and maintained at 50°C. 0.2 ml of SM buffer plus 10 µl of E. coli are in small tubes. Appropriate amounts (e.g., 1, 10, 100 µl) of each appropriate dilution are added to the bacteria. The tubes are placed on the 37°C wheel for 20-30 min. 3 ml of top agar are added to the phage and bacteria; the tube is vortexed for 10 sec, and poured onto a warm agar plate. Distribute the top agar evenly on the plate while rocking (rotating, swirling) by hand. Allow the top agar to harden at room temperature (approximately 10 min). Invert the plates and place at 37°C overnight.

The next morning look at the the plaques. They are usually of a reasonable size to count. However, if the plaques are very small (which does occur with the Olson lambdas), leave the plates at 37°C and check the size of the plaques hourly. Count the plaques on plates where the number is between 50 and 300. A mini mum of 100 is needed for reasonable statistical reliability; a plate with over 200 plaques may yield an incorrectly low number, as plaques overlap. How much accurracy you need is up to you. Calculate the titer of the stocks.

8- With the preparation of titered phage stocks derived from a single plaque, all the reagents are in hard to prepare lambda DNA. In addition to titered phage stocks, you need large (150-mm) NZY agarose plates, NZY top agarose, and E. coli C600 in 0.01 M magnesium sulfate. We switch from agar to (the much more ex pensive) agarose to avoid the umwanted impurities present in agar. We usually use two 150-mm plates for growth of each original phage plaque. The large NZY agarose plates are equilibrated at 37°C. NZY top agarose is at 50°C. Place 0.2 ml of SM buffer in a small tube and add 20 µl of E. coli (double the amount for a small plate). For poor-growing lambda, such as the Olson recombinants, we add 50,000 pfu to the tube. However, this number can vary greatly: higher for very poor -growing phage and much lower for phage which grow well. You may have to test several phage inputs. The goal is to achieve confluent lysis, maximum phage pro duction, without blowing away the E. coli prematurely. Add the lambda to the bacteria and SM buffer and place the tube on the 37°C wheel for 20-30 min. Add 5 ml (an increased amount for the large plates) of top agarose; vortex for 10 sec, and pour onto the warm 150-mm plate. Rotate (rock, swirl) the plate by hand to achieve an even distribution of top agarose. Allow the top agarose to harden (approximately 10 min) and invert the plate at 37°C overnight.

The next morning the plates should show confluent lysis. If individual plaques are visible, you may not achieve a usable amount of phage DNA. Start over at a higher input. We find that two confluently-lysed large plates will yield adequate amounts of DNA, even for poorly-growing lambdas.

9- Add 8 ml of SM buffer to each plate. Make sure the buffer covers the entire plate. Sterile conditions are no longer necessary, but neatness always counts. Rotate gently (or rock gently) at room temperature for 1 to 2 hr. The phage will elute into the buffer.

Decant (pipette) the buffer into a 15-ml conical tube (or equivalent): 5-6 ml/plate. Two large plates of the same phage are combined, as appropriate. Centrifuge the tube in an SS34 rotor with adaptors, RC-5B centrifuge (or equivalent) for 8 min at 4 K rpm (or equivalent). The debris will pellet, and most of the phage will reamain in the supernatant. Decant the supernatant (9-12 ml) to an Oak Ridge tube.

10- In this next , crucial step, DNase I is added to digest contaminating E. coli DNA. Lambda DNA, inside the phage particle, is protected from digestion. The integrity of the phage requires the presence of magnesium ions, and DNase activity requires magnesium ions, which are provided in SM buffer. Add 0.1 ml of DNase I (100 µg per ml; RNase-free) and mix gently. Place the tube at 37°C for 1-2 hr. (No attempt is made to remove RNA, and, in fact, RNA is used as a carrier for the DNA.)

11- Inactivate DNase I activity and disrupt the phage particles by the additions of 0.5 ml of 0.5 M EDTA, pH 8, and 0.5 ml of 10% SDS. Mix gently. (The removal of magnesium ions by the EDTA also causes the polysomes and ribosomes to fall apart. This helps during phage DNA purification.)

12- To remove protein and SDS, add 10 ml of phenol (previously equilibrated with TE buffer). Vortex hard for 20 sec. Centrifuge in an SS34 rotor, RC-5B centrifuge (or equivalent), for 10 min at 10 K rpm at 10°C. Decant the upper, aqueous phase to a fresh Oak Ridge tube. (We use an inverted 10 ml pipette without cot ton plug to transfer.) Add 10 ml of chloroform. Vortex hard for 20 sec. Centri fuge in an SS34, RC-5B centrifuge (or equivalent), for 10 min at 10 K rpm at 10°C. Decant the upper, aqueous phase to a fresh Oak Ridge tube (again we use an in verted 10 ml pipette). Note the volume transferred (usually around 10 ml).

13- Concentrate the DNA, and remove traces of phenol and chloroform, by alcohol precipitation. Add two-volumes of cold (95-100%) ethanol. Vortex for 10 sec to mix. Centrifuge in an SS34 rotor, RC-5B centrifuge (or equivalent), for at least 30 min at 12 K rpm at 10°C. Gently decant the supernatant. (We pour the alcohol off carefully while watching that the precipitate does not move.) To remove ex cess salt, gently add 10 ml of cold 70% ethanol. Do not disturb the nucleic acid precipitate. Centrifuge for 10 min (or more) at 12 K rpm at 10°C. Gently decant the supernatant. Add 10 ml of cold (95-100%) ethanol. Centrifuge for 10 min utes at 12 K rpm at 10°C. Gently decant the supernatant. Drain briefly and air dry briefly. Our experience with the Olson lambda DNAs is that the amount of precipitate is highly variable. Most of the precipitate is RNA.

14- Dissolve the precipitate in 2.5 ml of TE. This process may take longer than 1 hr at room temperature. Add 0.1 ml RNase (1 mg/ml, DNase free) and mix gently. (At this step, the contaminating RNA is removed by digestion). Place at 37°C for 1-2 hr. Add 0.1 ml of proteinase K (1 mg/ml, nuclease-free) and incubate at 37°C for 1-2 hr. Contaminating protein, including RNase and proteinase K itself, is re moved by digestion. Cool to room temperature.

15- Add 2.5 ml of phenol:chloroform (1:1; the phenol has previously been equili brated with TE buffer). Vortex hard for 10 sec. Centrifuge for 10 min at 10 K rpm, 10°C, in an SS34, RC-5B centrifuge (or equivalent). Decant the upper, aque ous layer to a Centricon-30 (Amicon Co.).

16- The Centricon takes the place of alcohol precipitation and/or dialysis to change buffer and concentrate the DNA. The Centricon works annoyingly slowly, but the recoveries are excellent. Read the manufacturer's (Amicon Co.) instruc tions. The plastic that composes the Centricon is resistant to traces of phenol and chloroform but is easily damaged by isoamyl alcohol. Many recipies and commer cially available phenol:chloroform add isoamyl alcohol as an anti-foam reagent. Do not use those recipes/products, or do not use the Centricon.

We use a Centricon-30 spun at 5 K rpm in an SS34, RC-5B centrifuge at 10°C. Other rotor/centrifuge combinations may have a different maximum speed; check Amicon's specifications. The choice of 10°C, rather than 4°C, is to avoid icing-up.

Under these conditions, a 2.5 ml sample will pass through the filter in about 30-60 min. We load the sample completely, do three 2.5 ml TE washes, and then spin for an additional 2 hr to reach minimum volume (usually 50-60 µl).

17- For the Olson lambda DNAs, we use 7 µl of the DNA to double-digest and run on a 1% agarose gel in 0.5xTBE. If all has gone well, the restriction enzyme cleav age pattern matches Olson's.

Reagents:

Standard reagents for DNA isolation and manipulation include:

TE buffer: 0.01 M Tris, 0.001 M EDTA, pH 8.0; usually diluted from a 100x stock: 1.0 M Tris, 0.1 M EDTA, pH 8.0.

0.5 M EDTA, pH 8.0: EDTA is sold as the disodium salt. In solution, Na 2EDTA has a pH near 5 and a saturation limit around 0.2 M. To achieve 0.5 M, NaOH needs to be added in small aliquots.

10% SDS: 10 g of sodium dodecyl sulfate dissolved in water to a final volume of 100 ml.

Ethyl alcohol: 95 or 100% and 70%, both at -20°C.

Phenol: Standard commercial phenol is quite dirty by molecular biology standards and must not be used. Several companies sell re-distilled phenol of "nucleic acid" or "molecular biology" grade. We are using Amresco's saturated phenol, which is a liquid, and comes with a small bottle of TE buffer to be added to the phenol. We want buffer-saturated phenol. Add the buffer to the phenol and shake. Allow to stand overnight in a refrigerator. Use an amber bottle to avoid light. We want to avoid oxidation of the phenol to produce quinones, which are highly reactive and detrimental to DNA. A reminder: a saturated solution cannot be created in an hour. At saturation, the upper phase is excess buffer.

Chloroform (CHCl3): any brand at A.C.S. purity is fine.