isolation of bacterial DNA

Question 1

Bacteria contain (comparisons to eukaryotes)

Answer 1

in the order of 1000-fold less DNA in their cells, on average, than eukaryotic cells. This DNA is not contained in a membrane-bound organelle as is seen in eukaryotes but is free-floating in the cytoplasm.Only one chromosome is usually seen, and this is a closed circular molecule. There are no associated packaging proteins (histones), such as those seen in eukaryotes. These properties make it relatively quick and easy for the bacteria to replicate its DNA and also for us to isolate it.

Question 2

E.coli

Answer 2

They are grown in a nutrient liquid broth to a high density of cells/ml of liquid. A few ml of this liquid are then mixed with a solution containing Sodium Dodecyl Sulphate (SDS) and sodium citrate. SDS is a detergent which will dissolve lipids. The SDS weakens the E.coli cell walls, causing the cells to burst or lyse. The cell contents are spilled into the liquid. This releases the DNA into the liquid, but also all of the cellular proteins. This includes proteins called nucleases which act to degrade DNA. These proteases need to be deactivated, which is done by unfolding or denaturing the proteins by heating the solution at 60oC. The sodium citrate also helps to inactivate these enzymes by mopping up magnesium ions which are essential for their activity. After allowing it to cool, ethanol is added to the solution and a glass rod inserted. DNA is insoluble in ethanol and will come out of solution as thread-like structures. These can be collected by winding onto a glass rod inserted in the solution.

Question 3

Part A. Isolating DNA from Bacteria

Answer 3

Take 5ml of E.coli broth culture and pipette into a boiling tube: dispose of the pipette in the disinfectant.

2. Add 1ml of SDS/Sodium citrate solution to the tube containing the bacteria.
3. Mix this for 5 min by gently swirling the tube.
4. Incubate the tube in the 60oC water bath for 30 min.
5. After 30min, remove the tube containing the now lysed (burst) bacteria from the water bath and allow it to cool for 10 min.
6. Collect a tube of ethanol from the ice bucket at the top of the lab.
7. Lower the glass rod into the bacterial solution. One person should hold the tube at about shoulder height in one hand, so that you can see better, using the fingers to hold the rod to one side of the tube.
8. The other partner should, with a pipette, trickle 10 ml of the ice-cold ethanol down the wall of the tube so that it forms a layer over the bacterial solution. While doing this, white material should start to become visible at the interface between the ethanol and the bacterial solution. This is DNA and RNA.
9. For the person holding the tube, while having the rod in contact with the bottom of the tube, slowly rotate it (don’t swirl), avoiding touching the sides of the tube. Some of the white material will start to move with the rod.
10. Keep rotating the rod until white threads start to come out of the solution and attach to the rod. Spool these around the rod and try lifting it out of the tube. If threads are still attached to the rod, then you have isolated DNA.
11. Once you have isolated DNA, put the glass rod upside-down in empty glass test tube (i.e. the threads in the tube) and drip 1 ml deionised water onto the rod to wash the threads off and into the tube.
12. You now have bacterial DNA resuspended in water:

Question 4

Part B. Separate Isolated DNA by Agar Gel Electrophoresis.

Answer 4

. Take 10 l of your DNA suspension and place in a microcentrifuge tube.

2. Add to it 10 l sample loading dye (leave the tip in the microcentrifuge tube).
3. Give your sample to the demonstrator to be loaded onto the gel. MAKE SURE YOU SEE HOW THIS IS DONE.
4. Once all the samples have been loaded, the gel with be run at a constant voltage of 100 V for approximately 30 min or until the tracking dye is about ¾ way down the gel.
5. After electrophoresis, the gel will be placed in Fast Blast stain for 2-3 min followed by destaining in warmed water for 2 x 5 min.
6. After destaining you should be able to visualise the DNA bands in your sample.

Question 5

Part C. Measure Concentration of Isolated DNA

Answer 5

Take 1000 l of extracted DNA and add it to an Eppendorf tube.

2. Take 30 l extracted DNA from the Eppendorf tube and add 2970 l deionised water and measure the optical density (absorbance) at 260 nm (dilution factor of 100). Use deionised water as a blank.
3. Take 300 l extracted DNA from the Eppendorf tube and add 2900 l deionised water and measure the optical density (absorbance) at 260 nm (dilution factor of 10). Use deionised water as a blank.
4. Once you have obtained an O.D value for your sample, use the following equation to work out the concentration of ds DNA in your sample. (You can use either of the OD values you obtained as long as they are less than 2.0).

Beer Lambert Law: A260 = E260 x l x c

Where:
A260 = optical density absorbance at 260nm
E260 = extinction coefficient at 260nm
l = light path length in cm
c = concentration in mg/ml

Question 6

Q1. What can you say about the appearance, reactivity or stability of DNA based on what occurred during your experiment?

Answer 6

The DNA after this experiment forms white clump like string pieces.

Question 7

Q2. Glass rods are used in physics experiments involving static electricity. With this in mind, can you think of any reason why a glass rod works better in this experiment than a plastic one?

Answer 7

because the exposed ends of the glass rod have polar chemical groups on them.

Question 8

Q3. Apart from the Fast Blast stain that was used today, list other stains/methods of visualising DNA on agar gels.

Answer 8

Ethidium Bromide (EtBr)