Editing Phage DNA Isolation (section)

== 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&nbsp; 100 uL LB. (10<sup>2</sup> pfu/mL).<br> 

2. Add to&nbsp; 5 mL of fresh log-phase E. coli K12 bacteria, and incunate at RT for 15 min. 

3. Add the mixture&nbsp; to 250 mL of LB media and shake&nbsp; for 6 h&nbsp;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&nbsp;% 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 &amp; 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 (&gt;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>