λ噬菌体DNA的小量制备
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1. Grow a fresh overnight culture of lambda host cells (we use K803 for EMBL3) in LB or T-broth fortified with 10mM Mg++ .
2. Mix a small amount (1 to 20μl, aim for an M.O.I. of +0.01) of high titer phage lysate (e.g. from a plate lysate) with 500μl of fresh cells. Incubate for 15min at 37℃. During this step, phage are allowed to adhere to bacterial cells.
3. Add cells plus adhered phage to 10ml of T-broth (one liter contains 10g bactotryptone, 5g NaCl) containing 10mM Mg++ . Shake at 37℃ for 3-6hrs, until cell lysis. In the meantime, check to see that the cultures first become turbid before they clear. That is, the bacterial cells must reach a certain density before they are lysed. A control culture containing only 0.5μl cells (no phage) may be useful to monitor cell growth. Our experience suggests if the bacterial cultures clear too soon, DNA yields may be reduced. This problem can be overcome by using a more dilute phage stock.
4. After the cultures clear, add 100μl chloroform and continue to shake for an additional 10min.
5. Centrifuge the lysates at 7,000rpm for 10min (in a Sorvall SS34 rotor) to pellet cellular debris. Transfer the supernatant to a fresh tube. 17x100mm disposable polypropylene tubes (Falcon brand has pop-top cap) are convenient. These can be rinsed and re-used.
6. Add DNase I (final is 1μg/ml) and RNase AI (final is 10μg/ml). For stocks solutions of DNase and RNase each at 10mg/ml, add 1 ul DNase and 10μl RNase. Incubate 30min at 37℃. [Note: There is no need to use high quality or pre-boiled RNase at this stage.]
7. Add 0.59 g NaCl (final is 1M) and 1.0g of PEG-8000 (final 10%). Mix thoroughly until the salt and PEG are completely dissolved. It may be convenient at this stage to snugly cap each tube and place them on a rocker platform for about 15minutes.
8. Once dissolved, place the tubes on ice for at least 90min. Do not freeze. If desired, the tubes may be left on ice overnight.
9. Pellet the phage-PEG complex by centrifugation at 10,000 rpm for 15min at 4℃. Use of a swinging bucket rotor (HB-4) is convenient for this step because the pellet will be concentrated at the bottom of each tube. Discard the supernatants.
10. Add 500μl TMN (10 mM Tris pH 7.5, 10mM Mg++ , 100mM NaCl) and gradually resuspend the pellet by repeated pipetting. This may take some work. Transfer the suspended phage to a microfuge tube.
11. Extract twice with 1 volume of chloroform (containing no isoamyl alcohol!). This step removes some of the PEG while the phage are still intact.
12. Add an equal volume of phenol to burst phage and to begin removing phage proteins. Mix by inversion and spin in a microfuge for about 1-3min (until upper aqueous phase becomes clear). Transfer the aqueous phase to a fresh microfuge tube.
13. Repeat step 12, using a 1:1 mixture of phenol:chloroform.
14. Repeat once again using a 24:1 mixture of chloroform:isoamyl alcohol.
15. Precipitate the phage DNA by adding 1/10 volume (perhaps about 40μl of 3M NaOAc, then 1ml of ice cold absolute EtOH. A nucleic acid precipitate should be immediately visible, but place on ice for at least 10minutes.
16. Pellet the DNA precipitate by centrifuging for 2-10minutes in a microfuge. If DNA yields are high, centrifuge for less time. In any case, don't centrifuge for too long.
17. Wash the pellet with 200μl of 70% EtOH, vortex briefly, then centrifuge briefly. Repeat this step once more.
18. Dry the pellet by leaving on the bench top, or by placing the sample in the SpeedVac concentrator for 5-10min.
19. Add 50-100μl TE (10mM Tris, pH 8.0, 1mM EDTA) and allow the pellet to resuspend slowly (overnight if possible). It may be useful to heat the sample at 65℃ for a few minutes.
20. Check the quality of the DNA by electrophoresis using a sample of about 1μl. I also like to check the sample for RNA contamination. I like to estimate DNA concentration (and total yield) using a Flurometer. Typical yields are about 5-10μg of DNA per 10ml culture.
Note :
This procedure can be scaled up proportionately for larger cultures, as desired. With larger volumes, polypropylene Oakridge tubes can be used in place of the 17x100mm polypropylene tubes.
2. Mix a small amount (1 to 20μl, aim for an M.O.I. of +0.01) of high titer phage lysate (e.g. from a plate lysate) with 500μl of fresh cells. Incubate for 15min at 37℃. During this step, phage are allowed to adhere to bacterial cells.
3. Add cells plus adhered phage to 10ml of T-broth (one liter contains 10g bactotryptone, 5g NaCl) containing 10mM Mg++ . Shake at 37℃ for 3-6hrs, until cell lysis. In the meantime, check to see that the cultures first become turbid before they clear. That is, the bacterial cells must reach a certain density before they are lysed. A control culture containing only 0.5μl cells (no phage) may be useful to monitor cell growth. Our experience suggests if the bacterial cultures clear too soon, DNA yields may be reduced. This problem can be overcome by using a more dilute phage stock.
4. After the cultures clear, add 100μl chloroform and continue to shake for an additional 10min.
5. Centrifuge the lysates at 7,000rpm for 10min (in a Sorvall SS34 rotor) to pellet cellular debris. Transfer the supernatant to a fresh tube. 17x100mm disposable polypropylene tubes (Falcon brand has pop-top cap) are convenient. These can be rinsed and re-used.
6. Add DNase I (final is 1μg/ml) and RNase AI (final is 10μg/ml). For stocks solutions of DNase and RNase each at 10mg/ml, add 1 ul DNase and 10μl RNase. Incubate 30min at 37℃. [Note: There is no need to use high quality or pre-boiled RNase at this stage.]
7. Add 0.59 g NaCl (final is 1M) and 1.0g of PEG-8000 (final 10%). Mix thoroughly until the salt and PEG are completely dissolved. It may be convenient at this stage to snugly cap each tube and place them on a rocker platform for about 15minutes.
8. Once dissolved, place the tubes on ice for at least 90min. Do not freeze. If desired, the tubes may be left on ice overnight.
9. Pellet the phage-PEG complex by centrifugation at 10,000 rpm for 15min at 4℃. Use of a swinging bucket rotor (HB-4) is convenient for this step because the pellet will be concentrated at the bottom of each tube. Discard the supernatants.
10. Add 500μl TMN (10 mM Tris pH 7.5, 10mM Mg++ , 100mM NaCl) and gradually resuspend the pellet by repeated pipetting. This may take some work. Transfer the suspended phage to a microfuge tube.
11. Extract twice with 1 volume of chloroform (containing no isoamyl alcohol!). This step removes some of the PEG while the phage are still intact.
12. Add an equal volume of phenol to burst phage and to begin removing phage proteins. Mix by inversion and spin in a microfuge for about 1-3min (until upper aqueous phase becomes clear). Transfer the aqueous phase to a fresh microfuge tube.
13. Repeat step 12, using a 1:1 mixture of phenol:chloroform.
14. Repeat once again using a 24:1 mixture of chloroform:isoamyl alcohol.
15. Precipitate the phage DNA by adding 1/10 volume (perhaps about 40μl of 3M NaOAc, then 1ml of ice cold absolute EtOH. A nucleic acid precipitate should be immediately visible, but place on ice for at least 10minutes.
16. Pellet the DNA precipitate by centrifuging for 2-10minutes in a microfuge. If DNA yields are high, centrifuge for less time. In any case, don't centrifuge for too long.
17. Wash the pellet with 200μl of 70% EtOH, vortex briefly, then centrifuge briefly. Repeat this step once more.
18. Dry the pellet by leaving on the bench top, or by placing the sample in the SpeedVac concentrator for 5-10min.
19. Add 50-100μl TE (10mM Tris, pH 8.0, 1mM EDTA) and allow the pellet to resuspend slowly (overnight if possible). It may be useful to heat the sample at 65℃ for a few minutes.
20. Check the quality of the DNA by electrophoresis using a sample of about 1μl. I also like to check the sample for RNA contamination. I like to estimate DNA concentration (and total yield) using a Flurometer. Typical yields are about 5-10μg of DNA per 10ml culture.
Note :
This procedure can be scaled up proportionately for larger cultures, as desired. With larger volumes, polypropylene Oakridge tubes can be used in place of the 17x100mm polypropylene tubes.
