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Phage DNA Isolation
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== ATCC Protocol<br> == To recover phage from freeze‑dried or frozen vial:<br> <br>a. Prepare an actively growing broth culture of the recommended host strain before opening the phage specimen. The host should be 18‑24 hours old.<br>b. Add approximately 1.0 ml of the recommended broth to a freeze-dried phage vial, 0.5 ml to a liquid cryovial.<br>c. Pre‑warm plates of the recommended medium in an incubator. Overlay the surface with 2.5 ml of melted 0.5% agar (same medium) which contains one or two drops of the 18‑24 hour host. The soft agar should be maintained at 43-45 oC until ready to pour. It may be advisable to use a water bath. Allow overlay to harden.<br>d. The re-hydrated phage can be serially diluted by passing 0.1 ml of the phage into a tube containing 0.9 ml of the broth medium. Repeat for as many passages as desired.<br>e. One drop of each dilution is spotted on the surface of the prepared plates. Allow to dry. Three to four dilutions can be placed on each plate. After overnight incubation, lysis should be visible. At the higher dilutions, individual plaques should be countable. <br>f. Many strains may also be titrated without a soft‑agar overlay. Pipette approximately 1.0 ml of the host onto the surface of each plate. After tilting the plate to ensure the entire surface is covered, the excess liquid is aspirated off. After the surface dries, the various dilutions of the phage are dropped onto the surface as before.<br> NOTE: Spotting the phage on plates makes visualizing the lysis easier. If phage is added directly to soft‑agar before pouring plates, hazy or tiny plaques may be difficult to see. Resistant host bacteria may also mask plaque formation. To propagate phage:<br>a. Phage may be propagated by preparing plates with the soft‑agar/host overlay as above and covering the surface with approximately 0.5 ml of the concentrated phage. Alternatively, you may add the phage directly to the melted agar/host before pouring over the plates. For larger amounts, large-size T‑flasks can be prepared with the recommended agar, and approximately 12.0 ml of melted soft‑agar/host poured over the surface. Phage is then allowed to run over hardened surface. Phage may also be added directly to melted soft‑agar before pouring as described above.<br> a. also be added directly to melted soft‑agar before pouring as described above.<br>b. After 24 hours incubation, or lysis is observed, the soft agar is scraped off the surface of the agar plates. Centrifuge at about 1000 rpm for 25 minutes to sediment the cellular debris and agar. Conserve the supernatant.<br>c. This supernatant is passed through a .22 um Millipore filter and the filtrate stored at 4-8oC. Lysates should remain viable under refrigeration for long periods. They may also be frozen with or without cryoprotectant. If available, liquid nitrogen storage is the best method for long term storage. Most phage can also be freeze‑dried. We use double‑strength skim milk mixed half-and-half with the filtrate.<br> NOTE: Broth propagation methods may also be employed with most phage. Unless otherwise noted, ATCC uses the Adams agar‑overlay method as described in M. H. Adams' Bacteriophages (Interscience Publishers, Inc., New York, 1959) for routine phage production.<br> <br> === T4 Phage Amplification with E. coli K12<br> === <br>1. Suspend 1 tiny piece of T4 phage from ATCC stock in 100 uL LB. (10<sup>2</sup> pfu/mL).<br> 2. Add to 5 mL of fresh log-phase E. coli K12 bacteria, and incunate at RT for 15 min. 3. Add the mixture to 250 mL of LB media and shake for 6 h at 37°C 4. The LB media will turn turbid indicating cell lysis. 5. Centrifuge at 4000 rpm for 10 min to remove bacterial debris 6. Collect supernatant.<br><br> === Phage stock === 1. After infection, add 5 ml of chloroform per liter of medium, incubate at 4 oC for 1 hour to o/n 2. Spin down bacterial debris at 6000 rpm for 10 min. 3. Spin down phage by ultracentrifuge, 20,000 X g (25000 rpm) for 30 to 60 min, 4 oC. 4. Suspend phage in 1 to 5 ml TE, incubate at 4 oC for 12 to 24 hours 5. Spin again at 4000 X g for 20 min. 6. Collect supernatant containing T4 phag. Add glycerol to final conc of 10%, aliquot and store at -80 oC. <br> === Precipitate Phage by PEG/NaCl<br> === <br> 1. Spin down bacterial debris from 250 ml to 1 L culture at 6000 rpm for 5 min. Collect supernatant. 2. Add solid PEG8000 and NaCl to adjust final concentration: 0.5 M NaCl, 5~10 % w/v PEG.<br> 3. Incubate at 4 oC for 2 hours to o/n 4. Spin down phage 6000 to 12000 rpm for 10 min. 5. Resuspend pellet in 5 ml P1. <br> === Extract DNA from Phage<br> === <br> 1. After precipitate phage using PEG/NaCl, resupend phage with 5 ml P1 with RNAse, add 5 ul benzonase, incubate at 37 <sup>o</sup>C for 1 hour.<br> 2. Add 5 ml P2 (for plasmid maxiprep) , mix by votex, incubate for 10 min.<br> 3. Add 5 ml of P3 (for plasmid maxiprep).<br> 4. Incubate on ice for 2 min.<br> 5. Spin at 13000 rpm for 2 X 2 min to pellet the white precipitate.<br> 6. Collect supernatant to another tube. <br> 7. Add equal volume (~15 ml) of isopropanol. Incubate on ice for 5 min.<br> 8. Spin at 13000 rpm for 10 min to precipitate DNA.<br> 9. Wash pellet with 70% ethanol, add 1 ml 70% ethanol, scrape DNA from centrifuge wall and transfer to a microcentrifuge tube, spin to pellet the DNA <br> 10. Dissolve DNA in 500 ul TE.<br> 11. Add 500 ul phenol-chloroform, mix by votex. 12. Spin at 13000 rpm for 2 min. 13. Collect supernatant to another tube. 14. Add 500 ul isopropanol, incubate on ice for 5 min. 15. Spin at 13000 rpm for 10 min. 16. Decant liquid. Wash pellet with 100 ul 70% ethanol. Spin briefly, remove liquid. 17. Add 100 ul TE to dissolve DNA. 18. Check DNA quality with nanodrop, if you see a lot of RNA, run phage DNA on agarose gel and purify phage band using spin column. Elute DNA with 100 ul TE.<br> 19. Digest phage DNA with HindIII and run digested and undigested DNA on gel.<br> 2012/12/25: T4 Phage DNA after phenol-chl clean up. [[Image:T4 Phage DNA 2012 12 25.jpg]][[Image:T4 phage DNA.JPG]][[Image:T4 Phage DNA 2013 1 2.jpg]] [[Image:T4 phage DNA 2013 1 6.jpg]]<br> === Protocol 2<br> === <br> A Nuclease Free Procedure for the Purification of Bacteriophage lambda DNA and Other Single- and Double-Stranded Viral Nucleic Acids (footnote) This protocol describes the purification of lambda DNA from plate or liquid lysates, although plate lysates tend to yield more consistent results. Volumes ranging from 1 ml to 250 ml can be processed using the corresponding NUCLEOBOND AX cartridge. Bacterial nucleic acids are separated with a specially activated matrix. An approximate correlation between cartridge size, culture volume and the amount of DNA is given in the table below. Note: Before starting with the isolation of Lambda DNA prepare buffer T2. T2: The PEG/NaCl mixture should be dissolved in distilled water by stirring. Heating the solution may facilitate this process. The T2 buffer should be stored at 4C and used cold. T4:Dissolve precipitated SDS by warming to 40C before use, if necessary. NOTE: All volumes given in the protocol are minimum volumes. The yield of lambda DNA depends on several properties like E. coli strain, particular lambda vector, titer of the lambda lysate, efficiency of inoculation, and culture conditions. Procedure Type of Cartridge<br> AX 20 AX 100 AX 500<br> Maximum culture volume 10 ml 50 ml 250 ml<br> Maximum amount of lambda DNA 12 µg 60 µg 300 µg Equilibration of the extraction matrix: 1. Add CHCl3 (1 µl/ml) to the lysate, mix, and centrifuge (6000 rpm; 10 min; 4C). Transfer the supernatant to a clean tube. 2. Add 30 mg KCl/ml lysate in order to adjust a 0.4 M final concentration of KCl for the lysate. 3. Adjust the pH of the solution with "diluted" phosphoric acid (H3PO4)to pH 6.3. ATTENTION: phosphoric acid is a strong acid. For the preparation of diluted phosphoric acid add 3-4 drops to 2 ml of water. 4. Resuspend the extraction matrix 0.5g of 2.5g of 10g of in buffer L1 and mix thoroughly matrix w/ matrix w/ matrix w/ (~5 min.) Use for the given cartridges 5 ml L1 10 ml L1 30 ml L1 the following amounts: 5. Centrifuge (greater or equal to 6000 rpm; 5 min.; RT) and discard the supernatant. 6. Add the lysate to the extraction matrix and mix gently by inverting the tube (~10 min.) manually or with an end over end mixer. 7. Pellet the extraction matrix by centrifugation (5000 rpm; 5 min.; RT) and transfer the supernatant to a new tube. Attention: Repeat this step if residual extraction matrix is visible. Preparation of the lambda particles: 8. Add buffer T2 (4C), mix thoroughly, 3 ml 15 ml 75 ml and incubate on ice for 60 min. 9. Centrifuge at greater than or equal to yes yes yes 10,000 x g at 4C for 10 min. Discard the supernatant and drain the pellet by standing the tube in a tilted position for 3 min. 10. Resuspend the pellet by pipetting 0.5 ml 1.5 ml 5 ml buffer T3 several times along the wall of the tube. (note #2) 11. Add buffer T4, mix gently, and 0.5 ml 1.5 ml 5 ml incubate at 65C for 10 minutes. Following this, put on ice for 5 minutes. 12. Add buffer S3 (pre-cooled 4C), mix 0.5 ml 1.5 ml 5 ml immediately by inverting the the tube 6-8 times, and centrifuge (greater or equal to 12,000 x g) at 4C for 35 min. Isolation of lambda DNA: 13. Equilibrate a NUCLEOBOND AX cartridge 1 ml 3 ml 8 ml with buffer L2. (For single-stranded DNA viruses such as M13, substitute buffer L1 in this step) 14. Transfer the clear supernatant of yes yes yes step 12, immediately after centrifugation onto the cartridge. 15. Wash the contaminating substances 2 x 1 ml 2 x 2.5 ml 2 x 5 ml from the cartridge with buffer L3. (For single-stranded viruses, substitute buffer L2 in this step.) 16. Elute the DNA from the cartridge with 1 ml 2.5 ml 6 ml buffer L5. (note #3) 17. Add about 0.75 volumes iso-propanol 0.75 ml 1.9 ml 4.5 ml equilibrated to room temperature. 18. Collect precipitated lambda DNA by centrifugation at ³12,000 x g for 30 minutes and wash the pellet with 70% ethanol. <br>Buffer solutions: Storage: T2: 25% polyethylene glycol (PEG 6000), 2.5 M NaCl 4C T3: 50 mM Tris-HCl, 150 mM NaCl, 30 mM EDTA, pH 7.7 RT T4: 2% SDS RT S3: 2.8 M K-acetate, pH 5.2 RT L1: 100 mM Tris-phosphate, 400 mM KCl, pH 6.3, 15% Ethanol RT L2: 100 mM Tris-phosphate, 900 mM KCl, pH 6.3, 15% Ethanol RT L3: 100 mM Tris-phosphate, 1000 mM KCl, pH 6.3, 15% Ethanol RT L5: 100 mM Tris-phosphate, 1000 mM KCl, pH 8.5, 15% Ethanol RT To make your own buffers or for more information please refer to Buffer Preparation and Nucleobond AX Buffers. NOTE 1: This yield of DNA from this procedure is very titer dependent. In addition, the minimum titer for one virus may not specifically apply to another. For example, the minimum recommended titer for phage lambda is about 4-5 x 10^9 pfu/ml. At this level one can expect to achieve a maximum theoretical yield of 200-250 ng lambda DNA per ml of lysate. However, for a 3 Kb single-stranded phagemid, the minimum recommended titer would be about 1-2 x 10^11 pfu/ml. At this level one can expect to achieve a maximum theoretical yield of 150-300 ng phagemid DNA per ml of lysate. The calculations below for molecular weight use 330 daltons per nucleotide of single-stranded DNA and 660 daltons per base pair of double stranded DNA. Double-Stranded Lambda DNA Size 50 Kb; MW = 50000 x 660 = 3.3 x 10^7 g/mole or 3.3 x 10^13 µg/mole Thus, at a titer of 5 x 10^9 the maximum amount of DNA per ml that can be purified can be calculated as: (5 x 10^9 pfu/ml / 6.02 x 10^23 /mole) x (3.3 x 10^13 µg/mole) = 1.65 / 6.02 ..approx.. 0.27 µg/ml Single-Stranded Phagemid Size 3 Kb; MW = 3000 x 330 ..approx.. 1 x 10^6 g/mole or 1 x 10^12 µg/mole Thus, at a titer of 1 x 10^11 the maximum amount of DNA per ml can be calculated as: (1 x 10^11 pfu/ml / 6.02 x 10^23 /mole) x (1 x 10^12 µg/mole) = 1 / 6.02 ..approx.. 0.17 µg/ml NOTE 2: If the lambda DNA consistently exhibits some degree of partial degradation as a result of exposure to endonuclease A from the plating bacteria (as visualized by some smearing below the lambda DNA band on an agarose gel), then add proteinase K to a final concentration of 200 µg/ml to the incubation in step 11 above. NOTE 3: For high GC content DNA: elute with a buffer of 50% formamide, 1.0 M KCl, 15% EtOH & 0.1 Tris-phosphate, pH 8.5, heated to 60C before loading. For the recommended procedure for preparing this buffer refer to N5 and L5 Elution Buffer Preparation. Precipitate the DNA with an equal volume of 70% (not 0.7 vol) isopropanol at room temperature and centrifuge at high speed (>12,000 x g) at 4C. The DNA has to be washed with 70% isopropanol to remove most of the formamide. A second washing step with 70% ethanol can be performed. After a short drying step DNA can be dissolved in an appropriate buffer for further manipulations. <br> == Stanford Lambda DNA Protocol == 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. Phenol: Chloroform (1:1): Mix one volume of buffer-saturated phenol with the same volume of CHCl3 in an amber bottle. Shake and place in the refrigerator overnight. The upper phase is excess buffer. We make our own mixture, because Amresco's phenol: chloroform contains isoamyl alcohol as an anti-foam reagent, and isoamyl alcohol dissolves the plastic Centricon. For this procedure, the following reagents are required: 0.01 M MgSO4: used to resuspend E. coli C600. We use A.C.S. grade MgSO4·7H 2O. Sterilize by autoclaving or filtration. DNase I (free of RNase): Several companies sell this reagent. Our stock solu tion is 100 µg/ml in 0.01 M Tris, pH 8.0. We purchase DNAse I from Boehringer -Mannheim. The stock is aliquoted, frozen (-20°C), and thawed as needed. (no EDTA) Careful: DNAse I is very heat labile and is easily inactivated at room tem perature. RNAse (free of DNAse): We purchase this enzyme from Boehringer-Mannheim. (Even the smallest amount of contaminating DNAse will degrade your precious DNA.) The RNAse is aliquoted and frozen (-20°C). Specialized reagents for growing coliphage lambda: NZY-amine medium (called NZYM medium in the CSH book): per liter: NZ-amine (also known as "casein enzymatic hydrolysate"; e.g., Sigma cat.# C -0626) 10 g<br> Yeast extract 5 g<br> NaCl 5 g<br> MgSO4·7H2O 2 g Sterilize by autoclaving. For agar plates (100-mm plates used for counting or isolating plaques), add 15 g of agar per liter before autoclaving. For top agar, add 7 g of agar per liter before autoclaving. For ease of handling, we prepare top agar in 100-200 ml batches, rather than 1 l batches. For large (150-mm) agarose plates, used when growing lambda to prepare DNA, add 15 g of agarose per liter before autoclaving; top agarose requires 7 g of agarose per liter before autoclav ing. Unless you have an unlimited supply budget, you should use agarose ony where essential. We make both NZY-agar and NZY-agarose plates and top media to save money. Maltose (20%): Dissolve 20 g of maltose in a final volume of 100 ml. Sterilize by filtration. Maltose, as all sugars, tends to become caramel upon autoclaving. SM buffer: We do not consider gelatin to be a component of SM buffer. In this usage, we differ from the CSH book. Throughout our text, SM buffer is composed of: per liter: NaCl 5.8 g<br> MgSO4·7H2O 2 g<br> 1 M Tris, pH 7.5, 50 ml (The CSH book uses the nomenclature "Tris·HCl", which is not technically correct. We start with Tris·OH and add HCl to pH 7.5. Therefore, our Tris buffers do not have additional sodium ions. Incidentally, Tris·OH is half of the cost of Tris·HCl.) Gelatin (2%): We purchase a sterile 2% gelatin solution from Sigma (cat. # G -1393) or other supplier. It's inexpensive, and we use very little. You could save money by making this solution yourself. 2% gelatin is not quite a liquid at room temperature. We warm the bottle gently in a microwave oven just before use. Gelatin is added to SM buffer (5 ml/liter)when the combined buffer is used to stabilize phage. Throughout our text, when gelatin is to be added, we state so explicitly. <br> == Labda Phage<br> == Qiagen Lambda DNA purification kit (or equivalent)<br>Buffer L1:<br>20 mg/ml RNAse A, 6 mg/ml DNAse I, 0.2 mg/ml BSA, 10 mM EDTA, 100<br>mM Tris-HCl, 300 mM NaCl, pH 7.5 (on ice)<br>Buffer L2:<br>30% polyethylene glycol (PEG6000), 3 M NaCl (on ice)<br>Buffer L3:<br>100 mM Tris-HCl, 100 mM NaCl, 25 mM EDTA, pH 7.5<br>Buffer L4:<br>4% SDS<br>Buffer L5:<br>2.55 M potassium acetate, pH 4.8<br>Buffer QBT: 750 mM NaCl, 50 mM MOPS, 15% ethanol, pH 7.0, 0.15% Triton X-100<br>Buffer QC: 1.0 M NaCl, 50 mM MOPS, 15% ethanol, pH 7.0<br>Buffer QF:<br>1.25 M NaCl, 50 mM Tris, 15% ethanol, pH 8.5<br><br> Day 1<br>1) Prepare a 150 ml overnight phage lysate culture by mixing 0.5 ml of an overnight culture of E.coli B cells and 10^6 plaque purified phage. This is probably about 10 ul. Add this mixture to 150 ml of media. Grow it overnight at 37 C. Day 2<br>2) Using a glass pipette, add 140 ul of chloroform to the culture to kill the E. coli. Mix gently. Centrifuge the overnight culture at 3900 rpm (Beckman CPR centrifuge) or 5000 rpm (Beckman J2-HC centrifuge) for 5 minutes to pellet unlysed cells and debris. 3) Save the supernatant by pouring it into a flask. Discard the pellets. 4) Add 216 l of buffer L1 to the supernatant from step 3. Incubate at 37 C for 30 minutes. After the incubation, save 0.5 ml for later analysis. 5) Add 30 ml of ice cold buffer L2, mix gently, and incubate the sample on ice for 60 minutes. 6) Centrifuge at 6000 rpm for 30 minutes. Discard the supernatant by pouring it out. Drain the bottle/tubes by placing it/them upside down for 1 minute. Do not wipe the inside of the bottle/tubes. 7) Use a pipette to resuspend the pellet(s) in a total of 9 ml of buffer L3 (not 9 ml per tube).<br>Note that the pellets are probably difficult to see. Pool the solutions into one tube. 8) Add a total of 9 ml of buffer L4. Mix gently (the phages may break easily).<br>Note: If we are short of time, store the sample at 4 C (refrigerator). Day 3<br>9) Incubate the tubes at 70 C for 20 minutes, and then let them cool on ice. 10) Add a total of 9 ml of buffer L5. Mix gently but immediately by inverting the tube 4 to 6 times to mix it well. The solution will become cloudy and viscous. Centrifuge it at 11000 rpm at 4 C for 30 minutes. While the sample is centrifuging, start step 12. When the spin is finished, immediately remove and save the supernatant. Discard the pellet. 11) If the solution is cloudy, centrifuge the supernatant again at 11000 rpm at 4 C for 10 minutes. Save the supernatant and discard the pellet.<br>Note: If we are short of time, store the sample at 4 C (refrigerator). 12) Equilibrate the Qiagen-tip 100 with 5 ml of buffer QBT. 13) Add the supernatant from step 11 to the column. Discard the flow-through. 14) Wash the column with 10 ml of buffer QC. Discard the flow-through. 15) Elute the DNA with 5 ml of buffer QF. Save the flow-through which contains your DNA or RNA.<br>Note: If time is short, the procedure can be stopped at any of the steps 15 through 19. 16) Precipitate the DNA by adding 0.7 volumes of isopropanol. This means that you measure the volume of your sample, multiply it by 0.7, and add that amount of isopropanol. Remember to also precipitate the aliquot saved on step 3. Note: If you stop here, put the sample in the refrigerator (4 ̊C). Day 4<br>17) Centrifuge for 30 minutes at 10000 rpm. Discard the supernatant. 18) Wash the pellet by adding 5 ml of 70% ethanol for the main sample, or 0.5 ml for the aliquot. Centrifuge at 10000 rpm for 10 minutes. Discard the supernatant again. 19) Add 5 ml 95% ethanol to the sample or 0.5 ml to the aliquot. Centrifuge at 10000 rpm for 10 minutes. Discard the supernatant, invert the tube, and let the pellet air dry for 5 or more minutes. When dry, you will not be able to smell the ethanol. 20) Add 100 l EB to the sample or 10 l EB (or AE) to the aliquot. 21) For the main sample, take 1 l of the nucleic acid from the previous step and read the A260 and A280 in the Nanodrop spectrophotometer. The spectrophotometer will calculate a concentration, assuming it is dsDNA.<br> <br> == Qiagen Column Purification<br> == === Buffers<br> === Qiagen Lambda DNA purification kit (or equivalent)<br>'''Buffer L1''': 20 mg/ml RNAse A, 6 mg/ml DNAse I, 0.2 mg/ml BSA, 10 mM EDTA, 100 mM Tris-HCl, 300 mM NaCl, pH 7.5 (on ice)<br>'''Buffer L2''': 30% polyethylene glycol (PEG6000), 3 M NaCl (on ice)<br>'''Buffer L3''': 100 mM Tris-HCl, 100 mM NaCl, 25 mM EDTA, pH 7.5<br>'''Buffer L4''': 4% SDS<br>'''Buffer L5''': 2.55 M potassium acetate, pH 4.8<br>'''Buffer QBT''': 750 mM NaCl, 50 mM MOPS, 15% ethanol, pH 7.0, 0.15% Triton X-100<br>'''Buffer QC''': 1.0 M NaCl, 50 mM MOPS, 15% ethanol, pH 7.0<br>'''Buffer QF''': 1.25 M NaCl, 50 mM Tris, 15% ethanol, pH 8.5<br> <br> === Procedure<br> === '''Day 1'''<br>1) Prepare a 150 ml overnight phage lysate culture by mixing 0.5 ml of an overnight culture of E.coli B cells and 10<sup>6</sup> plaque purified phage. This is probably about 10 μl. Add this mixture to 150 ml of media. Grow it overnight at 37 C.<br><br>Purification<br>'''Day 2'''<br>2) Using a glass pipette, add 140 ul of chloroform to the culture to kill the E. coli. Mix gently. Centrifuge the overnight culture at 3900 rpm (Beckman CPR centrifuge) or 5000 rpm (Beckman J2-HC centrifuge) for 5 minutes to pellet unlysed cells and debris.<br> <br>3) Save the supernatant by pouring it into a flask. Discard the pellets.<br><br> 4) Add 216 μl of buffer L1 to the supernatant from step 3. Incubate at 37 C for 30 minutes. After the incubation, save 0.5 ml for later analysis.<br><br> 5) Add 30 ml of ice cold buffer L2, mix gently, and incubate the sample on ice for 60 minutes.<br><br> 6) Centrifuge at 6000 rpm for 30 minutes. Discard the supernatant by pouring it out. Drain the bottle/tubes by placing it/them upside down for 1 minute. Do not wipe the inside of the bottle/tubes.<br> <br>7) Use a pipette to resuspend the pellet(s) in a total of 9 ml of buffer L3 (not 9 ml per tube).<br>Note that the pellets are probably difficult to see. Pool the solutions into one tube.<br><br> 8) Add a total of 9 ml of buffer L4. Mix gently (the phages may break easily).<br>Note: If we are short of time, store the sample at 4 C (refrigerator).<br><br> '''Day 3'''<br>9) Incubate the tubes at 70 C for 20 minutes, and then let them cool on ice.<br> <br>10) Add a total of 9 ml of buffer L5. Mix gently but immediately by inverting the tube 4 to 6 times to mix it well. The solution will become cloudy and viscous. Centrifuge it at 11000 rpm at 4 C for 30 minutes. While the sample is centrifuging, start step 12. When the spin is finished, immediately remove and save the supernatant. Discard the pellet.<br> <br>11) If the solution is cloudy, centrifuge the supernatant again at 11000 rpm at 4 C for 10 minutes. Save the supernatant and discard the pellet.<br>Note: If we are short of time, store the sample at 4 C (refrigerator).<br> <br>12) Equilibrate the Qiagen-tip 100 with 5 ml of buffer QBT.<br> <br>13) Add the supernatant from step 11 to the column. Discard the flow-through.<br> <br>14) Wash the column with 10 ml of buffer QC. Discard the flow-through.<br> <br>15) Elute the DNA with 5 ml of buffer QF. Save the flow-through which contains your DNA or RNA.<br>Note: If time is short, the procedure can be stopped at any of the steps 15 through 19.<br> <br>16) Precipitate the DNA by adding 0.7 volumes of isopropanol. This means that you measure the volume of your sample, multiply it by 0.7, and add that amount of isopropanol. Remember to also precipitate the aliquot saved on step 3.<br> <br>Note: If you stop here, put the sample in the refrigerator (4 ̊C).<br><br> '''Day 4'''<br>17) Centrifuge for 30 minutes at 10000 rpm. Discard the supernatant.<br><br> 18) Wash the pellet by adding 5 ml of 70% ethanol for the main sample, or 0.5 ml for the aliquot.<br>Centrifuge at 10000 rpm for 10 minutes. Discard the supernatant again.<br><br> 19) Add 5 ml 95% ethanol to the sample or 0.5 ml to the aliquot. Centrifuge at 10000 rpm for 10 minutes. Discard the supernatant, invert the tube, and let the pellet air dry for 5 or more minutes. When dry, you will not be able to smell the ethanol.<br> <br>20) Add 100 l EB to the sample or 10 l EB (or AE) to the aliquot.<br><br> 21) For the main sample, take 1 l of the nucleic acid from the previous step and read the A 260 and A 280 in the Nanodrop spectrophotometer. The spectrophotometer will calculate a concentration, assuming it is dsDNA.<br> <br>The general rule is that if the phage contains DNA, an absorbance at 260 nm of 1.0 equals a concentration of 50 g/ml. Back calculate how much DNA is in your sample.<br> == <br>AX Column Purification<br> == <br>T2: 25% polyethylene glycol (PEG 6000), 2.5 M NaCl 4C<br>T3: 50 mM Tris-HCl, 150 mM NaCl, 30 mM EDTA, pH 7.7 RT<br>T4: 2% SDS RT<br>S3: 2.8 M K-acetate, pH 5.2 RT<br>L1: 100 mM Tris-phosphate, 400 mM KCl, pH 6.3, 15% Ethanol RT<br>L2: 100 mM Tris-phosphate, 900 mM KCl, pH 6.3, 15% Ethanol RT<br>L3: 100 mM Tris-phosphate, 1000 mM KCl, pH 6.3, 15% Ethanol RT<br>L5: 100 mM Tris-phosphate, 1000 mM KCl, pH 8.5, 15% Ethanol RT<br> <br> Procedure <br> Type of Cartridge<br> AX 20 AX 100 AX 500<br> Maximum culture volume 10 ml 50 ml 250 ml<br> Maximum amount of lambda DNA 12 µg 60 µg 300 µg<br>'''Equilibration of the extraction matrix:'''<br>1. Add CHCl3 (1 µl/ml) to the lysate, mix, and centrifuge (6000 rpm; 10 min; 4C). Transfer the supernatant to a clean tube. <br>2. Add 30 mg KCl/ml lysate in order to adjust a 0.4 M final concentration of KCl for the lysate. <br>3. Adjust the pH of the solution with '''"diluted"''' phosphoric acid (H3PO4)to pH 6.3. '''ATTENTION:''' phosphoric acid is a strong acid. For the preparation of diluted phosphoric acid add 3-4 drops to 2 ml of water. <br>4. Resuspend the extraction matrix in buffer L1 and mix thoroughly (~5 min.) Use for the given cartridges the following amounts: (0.5g of matrix w 5 ml L1 / 2.5g of matrix w 10 ml L1 / 10g of matrix w 30 ml L1)<br> <br>5. Centrifuge (greater or equal to 6000 rpm; 5 min.; RT) and discard the supernatant. <br>6. Add the lysate to the extraction matrix and mix gently by inverting the tube (~10 min.) manually or with an end over <br>end mixer. <br>7. Pellet the extraction matrix by centrifugation (5000 rpm; 5 min.; RT) and transfer the supernatant to a new tube. <br>'''Attention:''' Repeat this step if residual extraction matrix is visible. <br>'''Preparation of the lambda particles:'''<br>8. Add buffer T2 (3 ml /15 ml/ 75 ml) (4 oC), mix thoroughly, and incubate on ice for 60 min. <br>9. Centrifuge at greater than or equal to 10,000 x g at 4C for 10 min. Discard the supernatant and drain the pellet by standing the tube in a tilted position for 3 min. (yes / yes / yes) <br>10. Resuspend the pellet by pipetting buffer T3 several times along the wall of the tube. ([http://www.nestgrp.com/protocols/nucleobond_ax/nuclease_free.html#note2 note #2]): (0.5 ml/ 1.5/ ml /5 ml ) <br>11. Add buffer T4, mix gently, and incubate at 65C for 10 minutes. Following this, put on ice for 5 minutes. (0.5 ml /1.5 ml/ 5 ml) <br>12. Add buffer S3 (pre-cooled 4C), mix immediately by inverting the the tube 6-8 times, and centrifuge (greater or equal to 12,000 x g) at 4C for 35 min. (0.5 ml/ 1.5 ml/ 5 ml) <br>'''Isolation of lambda DNA:'''<br>13. Equilibrate a NUCLEOBOND AX cartridge with buffer L2. (For single-stranded DNA viruses such as M13, substitute buffer L1 in this step) (1 ml/ 3 ml/ 8 ml) <br>14. Transfer the clear supernatant of step 12, immediately after centrifugation onto the cartridge. (yes/ yes/ yes) <br>15. Wash the contaminating substances from the cartridge with buffer L3. (For single-stranded viruses, substitute buffer <br>L2 in this step.) (2 x 1 ml /2 x 2.5 ml /2 x 5 ml) <br>16. Elute the DNA from the cartridge with buffer L5. ([http://www.nestgrp.com/protocols/nucleobond_ax/nuclease_free.html#note3 note #3]) (1 ml / 2.5 ml / 6 ml) <br>17. Add about 0.75 volumes iso-propanol equilibrated to room temperature. (0.75 ml / 1.9 ml / 4.5 ml) <br>18. Collect precipitated lambda DNA by centrifugation at 12,000 x g for 30 minutes and wash the pellet with 70% ethanol. <br> NOTE 1: This yield of DNA from this procedure is very titer dependent. In addition, the minimum titer for one virus may not specifically apply to another. For example, the minimum recommended titer for phage lambda is about 4-5 x 10^9 pfu/ml. At this level one can expect to achieve a maximum theoretical yield of 200-250 ng lambda DNA per ml of lysate. However, for a 3 Kb single-stranded phagemid, the minimum recommended titer would be about 1-2 x 10^11 pfu/ml. At this level one can expect to achieve a maximum theoretical yield of 150-300 ng phagemid DNA per ml of lysate. The calculations below for molecular weight use 330 daltons per nucleotide of single-stranded DNA and 660 daltons per base pair of double stranded DNA. Double-Stranded Lambda DNA Size 50 Kb; MW = 50000 x 660 = 3.3 x 10^7 g/mole or 3.3 x 10^13 µg/mole Thus, at a titer of 5 x 10^9 the maximum amount of DNA per ml that can be purified can be calculated as: (5 x 10^9 pfu/ml / 6.02 x 10^23 /mole) x (3.3 x 10^13 µg/mole) = 1.65 / 6.02 ..approx.. 0.27 µg/ml Single-Stranded Phagemid Size 3 Kb; MW = 3000 x 330 ..approx.. 1 x 10^6 g/mole or 1 x 10^12 µg/mole Thus, at a titer of 1 x 10^11 the maximum amount of DNA per ml can be calculated as: (1 x 10^11 pfu/ml / 6.02 x 10^23 /mole) x (1 x 10^12 µg/mole) = 1 / 6.02 ..approx.. 0.17 µg/ml NOTE 2: If the lambda DNA consistently exhibits some degree of partial degradation as a result of exposure to endonuclease A from the plating bacteria (as visualized by some smearing below the lambda DNA band on an agarose gel), then add proteinase K to a final concentration of 200 µg/ml to the incubation in step 11 above. NOTE 3: For high GC content DNA: elute with a buffer of 50% formamide, 1.0 M KCl, 15% EtOH & 0.1 Tris-phosphate, pH 8.5, heated to 60C before loading. For the recommended procedure for preparing this buffer refer to N5 and L5 Elution Buffer Preparation. Precipitate the DNA with an equal volume of 70% (not 0.7 vol) isopropanol at room temperature and centrifuge at high speed (>12,000 x g) at 4C. The DNA has to be washed with 70% isopropanol to remove most of the formamide. A second washing step with 70% ethanol can be performed. After a short drying step DNA can be dissolved in an appropriate buffer for further manipulations. <br> [[Category:Protocols]]
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