Extracting Cyanophage DNA from a Lysate

Protocol

Extracting Cyanophage DNA from a Lysate

Tucson High School

Biotechnology Course

Spring 2010

Summary: You will be extracting DNA from the viruses that you have isolated so that we can begin to use molecular techniques to investigate these viruses. DNA extraction involves separating viruses from cells, removing the DNA from the viral capsid, and cleaning up the DNA for use. (For time purposes, I have already begun this process and you will finish it)

1) Vortex your tube of purification resin for ~10 seconds and transfer 1 ml to a 2 ml microtube.

2) Get a virus sample from the ice bucket. Write your virus # in your notebook.

3) Pipet 500 µl of your virus sample into the tube with the purification resin. Mix by inverting the tube several times.

4) Remove the plunger from a sterile 3 ml syringe. Attach a minicolumn to the bottom of the syringe and add the contents of your 2 ml tube (contains purification resin and your virus) to the syringe.

5) Insert the plunger into the syringe and push the plunger so that the liquid from the slurry drips into your waste tube (the DNA remains on the column).

6) Remove the minicolumn from the syringe and then pull out the plunger.

7) Reattach the minicolumn to the syringe and add 2 ml 80% isopropanol to the syringe.

8) Insert the plunger into the syringe and push the plunger so that the isopropanol drips into your waste tube. (this washes the DNA on the column)

9) Remove the minicolumn from the syringe and place the minicolumn into a 1.5 ml microtube with your initials written on the side.

10) Centrifuge the minicolumn in the tube at 10,000xg for 2 minutes (removes all liquid remaining in the column).

11) Place the minicolumn into a new 1.5 ml microtube labeled with the virus # and your initials on the side of the tube. Discard the old tube.

12) Get TE buffer from the hot water bath and pipet 100 µl of this 80ºC TE Buffer on to the minicolumn. Put the cap over the top of the tube and immediately vortex for 1 minute.

13) Centrifuge at 10,000xg for 30 seconds (this elutes the phage DNA into your tube).

14) Repeat steps 12 and 13 with 50 µl of TE Buffer (get another tube of TE Buffer from the hot water bath). (this extra step ensures that you recover as much DNA as possible off of the minicolumn)

15) Discard the minicolumn.

16) Label a 500 µl microtube with your virus number and initials. Put 10 µl of your final product into this tube (I will use this to quantify the DNA you have recovered).

17) Close both of your tubes (make sure they are labeled!!) and put them into the ice bucket labeled “Final Samples”.