2. Gel Electrophoresis

3. Agarose Gel Electrophoresis Electro = flow of electricity, phoresis, from the Greek = to carry across is an analytical technique used to separate DNA, RNA and Protein fragments by size.DNA An electric field forces the fragments to migrate through a gel.electric fieldgel

4. DNA molecules normally migrate from negative to positive potential due to the net negative charge of the phosphate backbone of the DNA chain.phosphate Longer molecules migrate more slowly because they are more easily 'trapped' in the network.

5. DNA is negatively charged. +- Power DNA  When placed in an electrical field, DNA will migrate toward the positive pole (anode). HH O2O2 An agarose gel is used to slow the movement of DNA and separate by size. Scanning Electron Micrograph of Agarose Gel (1×1 µm)  Polymerized agarose is porous, allowing for the movement of DNA

6. +- Power DNA How fast will the DNA migrate? …gel electrophoresis separates DNA according to size small large Within an agarose gel, linear DNA migrate inversely proportional to the log10 of their molecular weight.

7. Gel Electrophoresis

8. What is needed? The types of gel most commonly used for DNA electrophoresis are Agarose

9. Agarose Agarose is a linear polymer polysaccharide extracted from seaweed. D-galactose 3,6-anhydro L-galactose Agarose was first used in biology when Robert Koch* used it as a culture medium for Tuberculosis bacteria in 1882

10. Agarose (for relatively long DNA molecules 200 to 50,000 base pairs long). Agarose is dissolved in buffer and heated, then cools to a gelatinous solid with a network of crosslinked molecules start end

11. An agarose gel is prepared by combining agarose powder and a buffer solution. polyacrylamidepolyacrylamide (for high resolution of short DNA molecules (Plasmid), for example in DNA sequencing) When sharper bands are required.DNA sequencing Agarose  Buffer  Flask for boiling 

12. AgaroseBuffer Solution Combine the agarose powder and buffer solution. Use a flask that is several times larger than the volume of buffer.

13. Agarose is insoluble at room temperature (left). The agarose solution is boiled until clear (right). *Be careful when boiling - the agarose solution may become superheated and may boil violently if it has been heated too long in a microwave oven. Melting the Agarose

14. Buffer - in this case TAE (TBE) The buffer provides ions in solution to ensure electrical conductivity. Tris-acetateTris-acetate buffer (TAE) usually at pH 8.0, and EDTA, which sequesters divalent cations. EDTA TAE has a lower buffer capacity than TBE and easily can become exhausted, but linear, double stranded DNA runs faster in TAETBE

15. Also needed are a power supply Gel chambers come in a variety of models, from commercial through home- made, and a variety of sizes

16. How does it work? DNA is an organic acid, and is negatively charged (remember, DNA for Negative) When the DNA is exposed to an electrical field, the particles migrate toward the positive electrode Smaller pieces of DNA can travel further in a given time than larger pieces

17. DNA negatively charged

18. Steps in running a gel DNA is prepared by digestion with restriction enzymes Agarose is made to an appropriate thickness (the higher the % agarose, the slower the big fragments run) and ‘melted’ in the microwave The gel chamber is set up, the ‘comb’ is inserted The agarose may have a DNA ‘dye’ added (or it may be stained later). The agarose is poured onto the gel block and cooled

19. DNA cut up using restriction endonucleases

21. Casting tray  Gel combs  Power supply  Gel tank  Cover Electrical leads  Electrophoresis Equipment

22. Gel casting tray & combs

23. Make up the gel which the DNA will be put into

27. The comb is removed, leaving little ‘wells’ and buffer is poured over the gel to cover it completely The DNA samples are mixed with a dense loading dye so they sink into their wells and can be seen

28. Dye added to the DNA  Bromophenol Blue + Sucrose

29. Buffer solution added to the tank

30. DNA samples loaded into wells

31. The DNA samples are put in the wells with a micropipette. Micropipettes have disposable tips and can accurately measure 1/1,000,000 of a liter

32. buffer  Add enough electrophoresis buffer to cover the gel to a depth of at least 1 mm. Make sure each well is filled with buffer.  Cathode (positive) Anode  (negative)  wells  DNA 

33. Place the cover on the electrophoresis chamber, connecting the electrical leads. Connect the electrical leads to the power supply. Be sure the leads are attached correctly - DNA migrates toward the cathode (red). When the power is turned on, bubbles should form on the electrodes in the electrophoresis chamber. Running the Gel

34. A gel being run Agarose block Positive electrode DNA loaded in wells in the agarose Black background To make loading wells easier Comb Buffer

35. Carefully place the pipette tip over a well and gently expel the sample. The sample should sink into the well. Be careful not to puncture the gel with the pipette tip.(5µlof sample +3µlof dye) Loading the Gel

36. Place the gel in the electrophoresis chamber.

37. Next? The power source is turned on and the gel is run. The time of the run depends upon the amount of current and % gel, and requires experimentation At the end of the run the gel is removed (it is actually quite stiff) The gel is then visualized - UV light causes the bands of DNA to fluoresce

38.  wells  Bromophenol Blue Cathode (-) Anode (+) Gel After the current is applied, make sure the Gel is running in the correct direction. Bromophenol blue will run in the same direction as the DNA. DNA (-) 

39.  100  200  300  1,650  1,000  500  850  650  400  12,000 bp  5,000  2,000 DNA Ladder Standard Inclusion of a DNA ladder (DNAs of know sizes) on the gel makes it easy to determine the sizes of unknown DNAs. - + DNA migration bromophenol blue 

40. Staining the Gel ***CAUTION! Ethidium bromide is a powerful mutagen and is moderately toxic. Gloves should be worn at all times. Ethidium bromide binds to DNA and fluoresces under UV light, allowing the visualization of DNA on a Gel. Ethidium bromide can be added to the gel and/or running buffer before the gel is run or the gel can be stained after it has run.

41. Staining the Gel Place the gel in the staining tray containing warm diluted stain. Allow the gel to stain for 25-30 minutes. To remove excess stain, allow the gel to destain in water. Replace water several times for efficient destain.

42. Ethidium Bromide requires an ultraviolet light source to visualize

43. Visualizing the DNA (ethidium bromide)  100  200  300  1,650  1,000  500  850  650  400  5,000 bp  2,000 DNA ladder  DNA ladder  PCR Product 1 2 3 4 5 6 7 8 wells  + - - + - + + - Samples # 1, 4, 6 & 7 were positive for DNA Primer dimers 

44.  250  1,500  1,000  500  750  2,000 bp DNA ladder  PCR Product wells  + - - - - + + - - + - + March 12, 2006 Samples # 1, 6, 7, 10 & 12 were positive for DNA

45. A gel as seen under UV light - some samples had 2 fragments of DNA, while others had none or one

46. DNA is stained using ethidium bromide

47. More…… Many samples can be run on one gel- but it is important to keep track Most gels have one lane as a ‘DNA ladder’ - DNA fragments of known size are used for comparison

48. Example Suppose you have a size standard with the following sized fragments: 1000 bp, 850 bp, 750 bp, 600 bp, 200 bp, 100 bp Size Standard 1000 bp 850 bp 750 bp 600 bp 200 bp 100 bp - + Sample 1Sample 2 Based this info, how big is the circled fragment? –850 bp

49. Still more…. The DNA band of interest can be cut out of the gel and the DNA extracted - Or DNA can be removed from the gel by Southern Blotting

50. References www.biotech.iastate.edu/publication/ppt- presentations Kreuzer, H., Massey, A., 2001, Recombinant DNA and Biotechnology,2nd ed. ASM Press, Washington Turner, P.C., et al, 1997, Instant Notes in Molecular Biology, Bios, Oxford Photos - L. D. Macdonald, 2003

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52. Prepare a Stock Solution of EDTA 0.5 M Na 2 EDTA (ethylenediamine tetraacetic acid) PH 8.0 18.6 gr EDTA 80 ml deionized water Adjust the PH with NaOH D/water up to 100 ml

53. Prepare a Stock Solution of TAE For 50 ml 0f 50X TAE buffer: 14.1gr Tris Base (2-amino-2-hydroxymethyl-propane-1,3-diol) 18.5 ml glacial acetic acid 5 ml EDTA D/W up to 50 ml Prepare a Working Solution of TAE For agarose gel electrophoresis, TAE can be used at a concentraion of 1x (1:50 dilution of the concentrated stock). Dilute the stock solution deionized water Finally 1 ml TAE add 49 ml D/W Be stored at room temperature

54. Prepare a Stock Solution of TAE for lab work for each group For 5ml 0f 50X TAE buffer: 1.41gr Tris Base (2-amino-2-hydroxymethyl-propane-1,3-diol) 1.85 ml glacial acetic acid 0.5 ml EDTA (0.186gr EDTA + 0.8ml D/Water adjust Ph to 8 by NaOH add D/water up to 1 ml) D/W up to 5 ml

55. Prepare Agarose Gelland Casting Tray 1-For a 2% agarose gel, weigh out 2g of agarose into a flask and add 100ml of 1 x TBE or TAE. 2-Heat solution in a microwave or boiling water bath until agarose is completely dissolved. 3-Allow to cool in a water bath set at 50 – 55°C for 10 min. 4-Prepare gel casting tray by sealing ends of gel chamber with tape or appropriate casting system. Place appropriate number of combs in gel tray.

56. Prepare of working solutionof TAE For agarose gel electrophoresis, TAE can be used at a concentration of 1x (1:50 dilution of the concentrated stock). Dilute the stock solution deionized water Finally 1 ml TAE add 49 ml D/W Be stored at room temperature

57. Prepare Agarose Gelland Casting Tray 5-cooled gel and pour into gel tray. Allow to cool for 15-30 min at room temperature. Gels can also be placed in a cold space and used the following day. 6-Remove comb(s), place in electrophoresis chamber and cover with buffer (TAE or TBE as used previously). 7- ((Add 5 ul of ethidium bromide 0.5 μg/ml working solution))

58. Loading Dye Adds loading dye (Bromophenol Blue25 mg,+ Sucrose4 g andH2O up to 10ml) to the DNA sample so that it will go into the 10- 20ul well (1 ul dye 1X :5 ul DNA) –makes it sink to the bottom It Adds blue color so you can see what you are pipetting

59. Loading DNA 1-Add loading buffer to samples. As a guideline, add 1.5 ul of 10x Loading Buffer to a 20-25 ul PCR/DNA solution. For more concentrated DNA solutions (e.g. plasmids), prepare tubes with 8 ul of 1x Loading Buffer and 2 ul of DNA. 2-Add 1:5, Loading dye:DNA 3-Load DNA and standard (Ladder) onto gel. 4-Electrophorese at 100V for 1 h. 5-Visualize DNA bands using UV lightbox or gel imaging system.

61. Purpose of Gel Electrophoresis A method for separating DNA Can be used to separate the size of –DNA –RNA –Protein We will be using it to separate DNA

62. DNA What you start with: A variety of different fragments of DNA all mixed together We will use gel electrophoresis to separate/sort these fragments

63. How It Separates The gel is a porous matrix (like a sponge) Separates DNA based on –Size –Charge

64. Separation by Size As DNA is moved through the gel, smaller sized fragments move through faster than larger sized fragments –Ex. A 100 base pair fragment will move through the gel faster than a 500 bp fragment start end Image taken without permission from http://www.dnai.org/b/index.html-- Gel Electrophoresis Animation http://www.dnai.org/b/index.html--

65. Separation Using Charge The charge on DNA is what makes it move through the gel DNA is a charged molecule. What is the charge on DNA? –Negative charge Why? –Phosphate group is negatively charged Image taken without permission from http://www.dnai.org/b/index.html-- Gel Electrophoresis Animation http://www.dnai.org/b/index.html--

66. Separation Using Charge The gel is hooked up to a power source DNA is loaded into the gel on the cathode (-) end Gel is placed in a buffer solution that will conduct electricity Electric current is run through the gel –DNA is attracted to the + end (anode) – “runs to the red” Image taken without permission from http://www.dnai.org/b/index.html-- Gel Electrophoresis Animation http://www.dnai.org/b/index.html--

67. The Gel Wells are created to put the DNA into We use agarose gels to separate DNA SIDE VIEW + - well TOP VIEW + - wells Direction DNA travels

68. Challenges DNA is colorless-- how will we see it on the gel & when we are loading it into the gel? How do we get the DNA to stay in the well (not float away)?

69. Solution #1 Problem #1: How can we see the DNA sample as we load it into the gel Problem #2: How can we make sure DNA won’t float away Solution: Add loading dye to the initial DNA sample!

70. Loading Dye Adds mass to the DNA sample so that it will go into the well –makes it sink to the bottom Adds blue color so you can see what you are pipetting

71. Solution #2 Problem: DNA is colorless. Once the DNA has been run through the gel, how can we see where it is on the gel? Solution: Add Ethidium Bromide (EtBr) to the gel

72. Ethidium Bromide The DNA intercalates with the Ethidium Bromide –Intercalates = inserts itself between bases EtBr will fluoresce under UV light

73. Relative Size vs. Absolute Size Looking at a gel, you can determine which fragments of DNA are bigger than others = Relative Size Which fragment is bigger, A or B? –Fragment A (didn’t travel as far in a fixed amount of time) A B (+) end (-) start

74. Absolute Size How can we determine the actual size of the DNA fragments (how many base pairs- bp)? Use a size standard –Also called a DNA ladder –Consists of a series of fragments of known sizes –Use it to compare to your DNA fragments

75. Example Suppose you have a size standard with the following sized fragments: 1000 bp, 850 bp, 750 bp, 600 bp, 200 bp, 100 bp Size Standard 1000 bp 850 bp 750 bp 600 bp 200 bp 100 bp - + Sample 1Sample 2 Based this info, how big is the circled fragment? –850 bp