Agarose gel electrophoresis: Agarose gel electrophoresis is a method used in biochemistry and molecular biology to separate a mixed population

of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge.Nucleic acid molecules are separated by applying an electric field to move the negatively charged molecules through an agarose matrix. Shorter molecules move faster and migrate farther than longer ones because shorter molecules migrate more easily through the pores of the gel. This phenomenon is called sieving. roteins are separated by charge in agarose because the pores of the gel are too large to sieve proteins. Procedure:
For a 1% agarose gel, weigh out 1g of agarose into a flask and add 100ml of 1 x TBE or TAE. There are a variety of agarose rodu!ts availa"le !ommer!ially #they have different melting ro erties$. For se arating sim le %&A fragments low melting tem erature agarose is ade'uate. (eat solution in a mi!rowave or "oiling water "ath until agarose is !om letely dissolved. Allow to !ool in a water "ath set at )0 * )) + for 10 min. ,re are gel !asting tray "y sealing ends of gel !ham"er with ta e or a ,la!e a ro riate num"er of !om"s in gel tray. ro riate !asting system.

Add ) ul of ethidium "romide to !ooled gel and our into gel tray. Allow to !ool for 1)-.0 min at room tem erature. /els !an also "e la!ed in a !old s a!e and used the following day.

0emove !om"#s$, la!e in ele!tro horesis !ham"er and !over with "uffer #TAE or TBE as used reviously$.

Add loading "uffer to sam les. 1oad %&A and standard #1adder$ onto gel. Ele!tro horesis at 1002 for 1 h. 2isuali3e %&A "ands using 42 light "ox or gel imaging system.

Reagents/Solutions:

water

)0 x TAE 5 676g Tris "ase, )8.1 ml of gla!ial A!eti! a!id, 100ml of 0.)9 E%TA # ( :.0$, 9ake u to 1 1 with water ) x TBE5 )7g Tris "ase5 68.)g of "ori! a!id5 60ml of 0.)9 E%TA # ( :.0$, 9ake u to 1 1 with

60 x ;odium Bori! A!id5 70.6g Borax Anhydrous, 9ake u to 1 1 with water 60< 1ithium Bori! A!id5 1ow-!ondu!tan!e %&A lithium "orate ele!tro horeti! media5 4; atent 8,1=.,=10 sold "y Faster Better 9edia 11+

Applications:

!stimation of the size of DNA molecules follo"ing restriction enzyme digestion, e.g. in restriction mapping of cloned DNA. Analysis of #R products, e.g. in molecular genetic diagnosis or genetic fingerprinting Separation of restricted genomic DNA prior to Southern transfer, or of RNA prior to Northern transfer.