S.Y.BSc SEMESTER III BOTANY PAPER II

1. S.Y.BSc SEMESTER IIIBOTANY PAPER II UNIT I: INSTRUMENTATION & TECHNIQUES CHROMATOGRAPHY BY Miss. SonamShukla Department of Botany SatishPradhanDnyanasadhana College, Thane (w)

2. Horizontal Gel Electrophoresis

3. Agarosegel electrophoresis is a method to separate DNA, or RNA molecules by size. • This is achieved by moving negatively charged nucleic acid molecules through an agarose matrix with an electric field (electrophoresis). • Shorter molecules move faster and migrate farther than longer ones .

4. At any given PH, exist in a solution as electrically charged species either as a cation (+) or anion(-). Under the influence of an electric field these charged particles will migrate either to cathode or anode, depending on the nature of their net charged

5. Electrophoresis is the movement of molecules by an electric current. • Nucleic acid moves from a negative to a positive pole.

6. • When placed in an electrical field, DNA will migrate toward the positive pole (anode). O2  H  DNA - + Power • DNA is negatively charged. • An agarose gel is used to slow the movement of DNA and separate by size.

7. Components of an Electrophoresis System • Power supply and chamber, a source of negatively charged particles with a cathode and anode • Buffer,a fluid mixture of water and ions • Agarose gel, a porous material that DNA migrates through • Gel casting materials • DNA ladder, mixture of DNA fragments of known lengths • Loading dye, contains a dense material and allows visualization of DNA migration • DNA Stain, allows visualizations of DNA fragments after electrophoresis

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

9. 1% agarose 2% agarose Agarose Gel • A porous material derived from red seaweed • Acts as a sieve for separating DNA fragments; smaller fragments travel faster than large fragments • Concentration affects DNA migration • Low conc. = larger pores better resolution of larger DNA fragments • High conc. = smaller pores better resolution of smaller DNA fragments

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

11. Melting the Agarose Agarose is insoluble at room temperature (left). The agarose solution is boiled until clear (right).

12. Pouring the gel

13. DNA buffer     wells Anode (positive) Cathode (negative)

14. Sample Preparation

15. Loading the Gel

16. Running the Gel

17. TAE (Tris-acetate-EDTA) and TBE (Tris-borate-EDTA) are the most common buffers for duplex DNA • Establish pH and provide ions to support conductivity • Concentration affects DNA migration • Use of water will produce no migraton • High buffer conc. could melt the agarose gel • New Sodium Borate (SB) buffer allows gels to be run at higher voltages in less time than traditional buffers Electrophoresis Buffer

18. Contains a dense substance, such as glycerol, to allow the sample to "fall" into the sample wells Contains one or two tracking dyes, which migrate in the gel and allow monitoring of how far the electrophoresis has proceeded. Loading Dye DNA samples are loaded into a gel AFTER the tank has been filled with buffer, covering the gel

19. Allows DNA visualization after gel electrophoresis • Ethidium Bromide • In gel staining DNA Staining

20. Analysis • After electrophoresis the gel is illuminated with an ultraviolet lamp to view the DNA bands. The ethidium bromidefluoresces reddish-orange in the presence of DNA. • photograph it with a digital camera.

23. Applications Estimation of the size of DNA molecules following restriction enzyme digestion, e.g. in restriction mapping of cloned DNA. Analysis of PCR products, e.g. in molecular genetic diagnosis or genetic fingerprinting Separation of DNA fragments for extraction and purification. Separation of restricted genomic DNA prior to Southern transfer, or of RNA prior to Northern transfer.

24. VERTICAL GEL ELECTROPHORESIS

25. To understand the principle of SDS-PAGE • To become familiar with the SDS-PAGE setup Experimental Goals

26. What is Electrophoresis? Electrophoresis is a laboratory technique for separating molecules based on their charge

27. The gel (matrix) itself is composed of either agarose or polyacrylamide. • Polyacrylamide is a cross-linked polymer of acrylamide. • Acrylamide is a potent neurotoxin and should be handled with care! The gel (matrix)

28. Have smaller pores than agarose, therefore high degree of resolving power. Can separate DNA fragments which range in size from 10-500 bp. DNA fragments which differ in size by one nucleotide can be separated from each other. Polyacrylamide gel electrophoresis is also used to separate protein molecules. Polyacrylamide gels

29. Separate proteins based on • Size (Molecular Weight - MW) • Allows us to • characterize • quantify • determine purity of sample • compare proteins from different sources Protein Electrophoresis

30. Proteins, unlike DNA, do not have a constant size to charge ratio • In an electric field, some will move to the positive and some to the negative pole, and some will not move because they are neutral • Native proteins may be put into gel systems and electrophoresed • An alternative to native protein gels forces all proteins to acquire the same size to charge ratio Protein Electrophoresis

31. SDS-PAGE(sodium dodecylsulphate-polyacrylamide gel electrophoresis) The purpose of this method is to separate proteins according to their size, and no other physical feature In order to understand how this works, we have to understand the two halves of the name: SDS and PAGE SDS-PAGE

32. SDS (sodium dodecyl sulfate) is a detergent that can dissolve hydrophobic molecules but also has a negative charge (sulfate) attached to it. If SDS is added to proteins, they will be soluablized by the detergent, plus all the proteins will be covered with many negative charges. Sodium Dodecylsulphate

33. PAGE is the preferred method for separation of proteins Gel prepared immediately before use by polymerization of acrylamide and N,N'-methylene bis acrylamide. Porosity controlled by proportions of the two components. Polyacrylamide Gel Electrophoresis (PAGE)

34. Polymerization of acrylamide is initiated by the addition of ammonium persulphate and the base N,N,N’,N’-tetramethylethylenediamine (TEMED) • TEMED catalyses the decomposition of the persulphate ion to give a free radical Catalyst of polymerization

35. Bis-Acrylamide polymerizes along with acrylamide forming cross-links between acrylamide chains Polyacrylamide Gels

36. Pore size in gels can be varied by varying the ratio of acrylamide to bis-acrylamide Polyacrylamide Gels • Protein separations typically use a 29:1 or 37.5:1 acrylamide to bis ratio

37. Side view

38. Movement of Proteins in Gel

39. Movement of Proteins in Gel • smaller proteins will move through the gel faster while larger proteins move at a slower pace

40. DC Power Source, Reservoir/Tank, Glass Plates, Spacers, and Combs • Support medium • Gel (Polyacrylamide) • Buffer System • High Buffer Capacity • Molecules to be separated • Proteins • Nucleic Acids Components of the System

41. Vertical Gel Format: Polyacrylamide Gel Electrophoresis Reservoir/Tank Power Supply Glass Plates, Spacers, and Combs

42. Step by Step Instructions on how to assemble the polyacrylamide gel apparatus

43. Prepare polyacrylamide gels Add diluted samples to the samplebuffer Heat to 95C for 4 minutes Load the samples onto polyacrylamide gel Run at 200 volts for 30-40 minutes Stain Procedure

44. Gel Preparation

46. Mix ingredients GENTLY! in the order shown above, ensuring no air bubbles form. Pour into glass plate assembly CAREFULLY. Overlay gel with isopropanol to ensure a flat surface and to exclude air. Wash off isopropanol with water after gel has set (+15 min). Gel Preparation

47. Tris buffer to provide appropriate pH SDS (sodium dodecyl sulphate)detergent to dissolve proteins and give them a negative charge Glycerol to make samples sink into wells Bromophenol Blue dye to visualize samples Heat to 95C for 4 minutes Sample Buffer

48. Run at 200 volts for 30-40 minutes Running Buffer, pH 8.3Tris Base       12.0 g Glycine          57.6 gSDS                4.0 g distilled water to 4 liter Loading Samples & Running the gel

49. SDS-PAGE

50. Staining Proteins in Gels • Coomassie Brilliant Blue • The CBB staining can detect about 1 µg of protein in a normal band. • Silver Staining • The silver stain system are about 100 times more sensitive, detecting about 10 ng of the protein.