EXPERIMENT TO DETERMINE MOLECULAR WEIGHT OF DNA USING AGAROSE GEL ELECTROPHORESIS
PRACTICAL DATE Friday 12th January 2018
SUBMISSION DATE 5:00 pm Friday 19th January 2018
This practical session will introduce you to the principles of agarose gel electrophoresis. You will set up a serial dilution of DNA which will be run on an agarose gel in order to estimate the molecular weight of the DNA molecules present.
Agarose gel electrophoresis (AGE):
In AGE, samples of nucleic acid are loaded onto a horizontal submerged slab of agarose gel in an electrophoresis tank, and an electric current is passed through the gel. Since DNA has an overall negative charge, the DNA molecules are attracted through the gel towards the positive electrode (the anode). Small fragments can pass easily through the pores of the gel whilst the movement of large fragments is retarded. Since DNA is of uniform diameter, the distance travelled through the gel by a particular DNA molecule is proportional to its molecular weight (or length in base pairs (bp)). A negatively-charged tracking dye is added to your samples to enable gel loading and to provide a visible front which moves down the gel under the influence of the current allowing the progress of electrophoresis to be monitored.
DNA can be visualised by staining the gel with an intercalating dye. These dyes integrate between the stacked base-pairs of double-stranded nucleic acids (intercalation); it is therefore taken up preferentially by the DNA within the gel. DNA bands within the gel can be seen by placing it on a transilluminator which provides a source of ultraviolet light. This makes the dye emit light (fluorescence), the amount of fluorescence is proportional to the amount of DNA (i.e. the brighter the band the more DNA is present).
Estimating molecular weight:
The distance travelled relates to the molecular weight of the DNA fragments (the molecular weight relates to the number of base pairs present in the molecule). In order to estimate the molecular weight of DNA a molecular weight marker (DNA ladder) is run alongside the samples. This contains a set of fragments of known molecular weight. The 100 bp ladder used in this experiment contains 14 fragments ranging from 100 bp to 3000 bp (see accompanying documentation on Blackboard). Comparing the distance travelled by the known fragments in the ladder to a DNA molecule allows us to estimate its molecular weight.
At the beginning of the practical session, details regarding health and safety (COSHH and Risk Assessments) will be given to you. Good laboratory practice must be adhered to at all times. In the course of this session, gloves must be worn at all times. Because of their affinity for DNA, intercalating dyes are potential mutagens. A commercial product “GelRed” (Biotium) which has low levels of toxicity is being used but should still be treated with care.
Work in pairs: Each pair requires:
1.5 ml Microfuge tubes.
P100 or P20, P10 micropipettes.
Yellow and white tips.
Water (200 µl).
100 bp ladder – Molecular weight markers (10 µl @ 5 ng/ µl).
Gel mould and electrophoresis tank.
Measuring cylinders (50 ml).
“Gel Red” DNA stain
Protocol 1 – Preparation of an Agarose Gel
1. The TAE buffer used to make and run your gel is held as a 50X stock i.e. it is 50 times more concentrated than required.
How much 50X TAE do we need to make 1L 1x working solution?
50X TAE ml
Total Volume 1000 ml
2. A 1.2 % (weight/volume) agarose gel will be prepared. How much agarose must be dissolved in 30 ml 1X TAE to get a 1.2 % gel?
3. Add the calculated amount of agarose to a flask and add 30 ml of 1x TAE. Mix by swirling.
4. Place your gel solution in the microwave for approx. 3 minutes at a Med High level – keep an eye on it so it doesn’t boil over.
NOTE: Take care when handling hot flasks. Microwaved solutions can get superheated and bubble over if shaken. Use heat resistant gloves and wear safety glasses.
5. Leave to cool for a few minutes before adding 1 l of “Gel Red” (an intercalating dye) and mixing. Pour the agarose into the gel mould and insert the comb to create the wells. Leave to set for about 20 minutes.
6. Once set, remove the comb and place the gel into the electrophoresis tank. Fill the tank with 1x TAE buffer. The gel is now ready to load.
Protocol 2 – Serial Dilution of DNA.
Label three microfuge tubes 1, 2 and 3.
Dispense 15 µl of H2O into each tube
Add 15 µl of the DNA stock (concentration 50 µg/µl) to tube 1. Use the vortex to mix (for about 10 sec) and centrifuge briefly (make sure your centrifuge is BALANCED) to ensure all liquid is at the bottom of the tube.
Remove 15 µl from tube 1 and add to tube 2. Mix thoroughly and centrifuge briefly to ensure all liquid is at the bottom of the tube.
Remove 15 µl from tube 2 and add to tube 3. Mix thoroughly and centrifuge briefly to ensure all liquid is at the bottom of the tube.
Protocol 3 – Agarose Gel Electrophoresis.
Label 3 microfuge tubes using a permanent marker.
Transfer 10 µl from tubes 1-3 that you set up in Protocol 2
Add 2 µl of tracking dye to each tube. Mix and centrifuge the tubes in the bench microfuge for a few seconds to ensure all the liquid is at the bottom of each tube.
Place your gel in the tank (note the direction of the electrophoresis – DNA will move towards the positive electrode), submerge your gel in 1X TAE Buffer.
Load 12 l of each sample onto the agarose gel:
Lane 1: Molecular weight marker
Lane 2: DNA – Tube 1
Lane 3: DNA – Tube 2
Lane 4: DNA – Tube 3
The gel will run at a voltage of 100V for 20-30 minutes. DNA molecules will migrate through the gel at a rate proportional to their molecular weight.
As your gel runs the intercalating dye that was added to the gel binds to your DNA. When the blue tracking dye has reached half way you can stop the run and transfer your gel to the transilluminator. The intercalating dye will fluoresce under UV light allowing you to visualise your DNA.
Complete the following tasks and write them up in a Word document. Please include your calibration curve from Excel and indicate where your unknown molecular weight sample lies on this curve.
1. Measure the distance each band of the Molecular Weight Markers has travelled from the well. Use Excel to plot molecular weight against distance travelled to generate a calibration curve. (15 marks)
2. Measure the distance your DNA sample has travelled from the well and use your calibration curve from the previous question to estimate the molecular weight of your DNA sample. (5 marks)
3. Describe the principles of gel electrophoresis and explain how it can be used to estimate molecular weight and quantify DNA within a sample. Remember to cite your sources correctly. (10 marks)
4. Evaluate the accuracy of your calibration curve and identify anything that might have affected it. (10 marks)