From DNA to Organism: A Study in DNA Function for the High School Biology Classroom
 
 

Modules 2: DNA and genes 

Procedures for Examining Plants' DNA for the Mutation of the AKT-1 Gene

     Part I. Getting out the goods

           Here, we use a commercially available kit to extract DNA from plants, the QLAGEN DNEASY Plant Mini-Kit.

           The first step in investigating the DNA of an organism is getting it out of the cells. Remember that the DNA of most organisms is kept in the nucleus of the cell so we need to open up both the cell and nuclear membranes to get at it. The biggest problem that we will face is the fact that the cell and the DNA inside of it is so small. We cannot just reach into the cell to get it. The other problem we will face is that there are a lot of chemicals in the cytoplasm that will breakdown the DNA if given the chance. Luckily scientists have figured out a way to get out just the DNA using a number of different chemicals. We will be using one such method today.

Follow the steps below. To help you keep track of where you are, check off the boxes as you go.

[ ]1.First we need to get some plant cells from which to get the DNA. Each lab  group has been provided with two plants to get their DNA from, one wild type (WT) and one mutant (MUT). For each of the plants we need to carefully obtain 200 mg (fresh weight) of leaves. To get them cut carefully at the base of the leaves and place them into a weighing dish. Make sure to label the two different samples.

WHY? The leaves are the easiest of the plant tissues to get DNA from.   Now we need to break apart the cell walls of the plants to get into the cells. To do this you will need to put your plant sample into a mortar and pestle. Allow your instructor to pour a small amount of liquid nitrogen into the mortar. Be careful not to splash the liquid nitrogen.

SAFETY WARNING: Make sure to keep your gloves and goggles on while you work with the liquid nitrogen. It is very cold and can be very dangerous. BE CAREFUL!

WHY? By putting the cells into a liquid nitrogen bath, we freeze them solid and then break them apart by mashing with the pestle. Try not to let your samples thaw out while you grind. It is normal for the liquid nitrogen to disappear while you mix; it is just evaporating to a gas!

[ ]2.When you finish your grinding, scrape your sample into a sample tube.   Make sure to label your tubes so you know which is which. To the tube add 400ml of the buffer labeled AP1. Also add 4ml of the RNase. Once these are added, mix the tubes.  

WHY?The buffer solution contains detergents that break apart the lipid of the cell membranes and also helps to keep a friendly environment for the DNA. The RNase will break down the RNA molecules found in the cell that we do not want to have in our final sample.      

[ ]3.Place your tubes into the hot water bath (should be at 65°C). They need to sit in this water bath for 10 minutes. Lift your tubes out to vortex mix them 2 or 3 times during the heating stage. 

WHY? The buffer and the heating help to break the cells apart more.


[ ]4.
Place your tubes into the hot water bath (should be at 65°C). They need to sit in this water bath for 10 minutes. Lift your tubes out to vortex mix them 2 or 3 times during the heating stage. 

WHY? The buffer and the heating help to break the cells apart more.

[ ]5.After the 10 minutes has passed, remove your tubes and add 130ml of the AP2 buffer. Mix the AP2 buffer into the solution and then place the tubes on ice for 5 minutes.

WHY? The AP2 buffer contains chemicals that will take out the detergents

After 5 minutes on ice, place the sample in the centrifuge for 5 minutes at high speed.
WHY? This step helps to pull the larger debris to the bottom of the tube, so we can get the DNA off the top.

[ ]6.Pipette the liquid off the top of the tube and place it into the purple sample tube supplied. Be careful to leave the solids at the bottom of the tube.   Centrifuge this tube for 2 minutes at high speed.

WHY? The purple column contains a filter that will pull out the largest of the debris in the solution.

[ ]7.Remove the top filter portion of the tube and throw it our. Pipette out the liquid from the bottom section of the tube and place it into a new sample tube. Be careful not to pipette out the white solid at the bottom of the tube. Place the liquid in a new sample tube. Again, make sure to label the tubes!

WHY? Our DNA, when in solution, is small enough to pass through the filter but too small to get stuck at the bottom of the tube during the spinning. By pulling off the solution off the top we are getting the majority of the DNA.

[ ]8.Add 750ml of the AP3 buffer to your new tube. Mix by GENTLY by pipetting the solution in and out.

WHY? The AP3 buffer contains ethanol which helps to precipitate (bring out of solution) the DNA so that we can isolate it from any other compounds that are still in the solution.

[ ]9.Take a DNA collection column (white) and sit it in a new, clean tube.   Take 650ml of your solution and add it to the top of the DNA collection tube. Centrifuge at high speed for 1 minute. After one minute, remove the column from the tube, pour out the liquid in the bottom of the tube and place the column back into the tube. Now put the remainder of your sample into the

WHY? By adding the DNA solution to the new collection tube we can pull it out of solution. The new column contains tiny silica (glass) beads that DNA will stick to, but little else does. This way the DNA will stick in the filter and any remaining debris will be washed through. Our DNA is now in the filter of the column.


[ ]10.Remove the old collection tube and put a new one on the bottom of your column. Rinse your DNA collection column by adding 500ml of AW buffer and centrifuging for 1 minute on high. Discard the liquid in the collection tube and add 500ml more and repeat the centrifuging.

WHY? This step is just used to clean up the DNA while it is still stuck on the beads. The DNA is still on the beads in the filter.

[ ]11.Throw away the old collection tube and place a new one under the DNA collection column. Add 100ml of the AE solution to the top of the tube. Let the solution sit at room temperature for 5 minutes and then centrifuge on high for 1 minute. Keep this solution in the collection tube.

WHY? Here we are dissolving the DNA in the AE solution and washing it off of the beads. At the end of this step, we will have our DNA in the solution in the collection tube, not in the filter.

[ ]12.Add 100ml more of the AE solution to the top of your tube and let it sit at room temp for 2 minutes. Centrifuge the solution on high for 1 minute. Keep the solution in the collection tube.

WHY? Here we are just washing once more to make sure that we can get all of the DNA out of the filter.

[ ]13.Discard the DNA collection column and keep the tube with the extracted DNA solution in it.

[ ]14.Using a marker, label the tube with your group’s initials and the sample ID, WT or MUT. Place these tubes into the freezer for storage until next class.

WHY? Freezing the DNA will preserve it until the next class when we will use it to analyze the mutant plant further.