Phage Miniprep Protocol
So you have to isolate phage DNA and dont want to spend hours... well, you could buy a magic merlin wizard phage miniprep kit, but you KNOW how I feel about that! I was sent the following protocol by a friendly usenet reader after having tried several methods with variable results. This produces enough DNA for subcloning and mapping the fragments in about 30 min.
You need to have 1.5 ml (or more) of a very high titre liquid lysate that has been cleared with chloroform. I will assume you have gotten that far. If not, refer to "Maniatis" or Current/Short Protocols in Molecular Biology (my favorite!) Good luck and feel free to email me if you have problems or further questions about this protocol. As always, standard disclaimers apply!
Reference: Santos, MA. (1991?). An improved method for small scale prep. of bacteriophage DNA based on phage precipitation by zinc chloride. Nucleic Acids Research 19:5442
TES: 0.1M Tris-HCl pH 8.0 0.1M EDTA 0.3% SDS
- Add DNAse and RNAse to a final concentration of 100 ug/ml and incubate at 37oC for 30 min.
- Add 20 ul of filter sterilized 2.0 M ZnCl2 to each 1 ml of lysate and incubate for 5 min at 37o
- Centrifuge 1 min and remove supernatant. There should be a substantial pellet containing the phage at the bottom of the tube.
- Resuspend the pellet in 500 ul of TES with the help of a tip or sterile toothpick and incubate at 65o for 15 min.
- Add 60 ul 3M potassium acetate pH 4.8 (standard alkaline lysis miniprep solution). Mix thoroughly and incubate on ice for 15-20 min.
- Centrifuge 1 min and remove supernatant to a new tube.
- Add an equal vol of isopropanol, mix and incubate on ice for 5 min.
- Centrifuge for 10 min, wash the pellet with 70% EtOH and dry. Resuspend in appropriate volume of water or TE
- I'm not sure if it is necessary to filter sterilize the ZnCl2.
- The ZnCl2 does "go bad" but I am at a loss as to what happens to it. I just know that after a month on the bench top, its ability to precipitate anything drops dramatically. Therefore, I make it up in small batches ie 1-5 ml. You only use 20 ul at a time!
- The very retentive may want to add a phenol:chloroform extraction between steps 6 and 7!