Prepared By: Mageswary Sivalingam

Transcription

1 Prepared By: Mageswary Sivalingam

2 A technique in which charged molecules are separated by their migration in an electric field. Applications - Gel electrophoresis is used in forensics, molecular biology, microbiology and biochemistry o o Estimation of the size of DNA molecules following restriction enzyme digestion Analysis of PCR products

3 To perform gel electrophoresis, 3 things are needed - Buffer system - Electric field - Medium

4 Purified from agar, gelatinous substance isolated from seaweed. Neutral, linear polysaccharide component Consist of repeating galactose and 3,6- anhydrogalactose subunits Agarose Subunit

5 Supporting media- porous media Commercially prepared agarose polymers are believed to contain ~800 galactose residues per chain Length varies from manufacturer to manufacturer Variability - gelling/ melting properties of agarose, sieving of molecules and recovery of molecule from the gel.

6 Agarose mixed with electrophoresis buffer - forms gel introduce molecular sieving effect Gelation formation of hydrogen bond pores. Low concentration produce gels with large pores Separation of molecules depend on agarose concentration and type of agarose

7 Range of separation of DNA fragments Type of Gel % (W/V) Range of DNA resolution of DNA(bp) Agarose Polyacrylamide

8 Dissimilar molecules migrates at different rates Net charge Highly charged migrate faster Molecular Size Smaller, compact molecule pass through more easily then the larger molecule - depend on number of base pairs

9 Shape of molecular globular molecules compared with fibrous molecule and - linear molecule compared with circular molecules migrate differently - Depend on agarose used and strength of applied current and also the strength of buffer

10 Concentration of Agarose Related to the properties of the gel - The size and shape of migrating molecules Applied Voltage - Mobility increases with incresing field strength limitation heating effects

11 Electrophoresis Buffer - Mobility depend on the ionic strength of the electrophoresis buffer - In the absence of ions eg. water, electric conductivity minimal - High ionic strength (10X buffer), electrical conductivity very efficient but significant heat generated - Tris Acetate EDTA (TAE) and Tris Borate EDTA (TBE)

12 Presence of EtBr in the gel - Decrease the negative charge of dsdna - Retard the migration rate

13 An electrophoresis chamber and power supply Gel casting trays Sample combs Electrophoresis buffer Gel sealing tape Microwave oven

14 Loading buffer - To give density - allow the sample to "fall" into the sample wells evenly and one or two - tracking dyes Ethidium bromide Transilluminator

15 Seal the edges of casting tray with gel sealing tape to form a mold Prepare sufficient electrophoresis buffer Heat the slurry in microwave oven until the Agarose dissolves Prepare solution of agarose in electrophoresis buffer at appropriate concentration

16 Fix the appropriate comb for forming the sample slot to the mold Pour warm agarose to the mold Mount the gel in the electrophoresis tank and add EB covering the gel Allow the gel to set and then remove the comb and the tape

17 Mix the sample with gel loading buffer Load the sample mixture into the slots Stain the completed gel with EtBr Apply the Voltage

18 View under UV transilluminator

19 Faint or no bands on the gel -insufficient quantity or concentration of DNA - DNA was degraded Smeared DNA bands - DNA was degraded - Too much DNA was loaded on the gel - Improper electrophoresis conditions were used - There was too much salt in the DNA

20 H David et al. Practical Skills in Biomolecular Sciences. 2 nd edition; Pearson Education Limited. Joseph Sambrook and David W. Russell; Molecular Cloning, 3rd edition.cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2001

21 THANK YOU