A. NAZLI BAŞAK

1. BIO 306.01/02 EXPERIMENT 4 Recovery of DNA from Agarose gels A. NAZLI BAŞAK

2. Recovery of Dna from Agarose Gel • Agorose gel e-phoresis is widely used in molecular biology labs; it is based on the principle that DNA molecules of different size or topology migrate with different velocities thru agorose gels when they are driven by an electrical field.

3. Recovery of Dna from Agarose Gel • Since DNA is charged negatively (phosphate backbone), it will migrate towards the (+) pole, anode, of the e-phoretic chamber according to its size and conformation. • This allows the separation of different molecules, which can then be stained by EtBr in order to make the DNA visible under UV-light. • The position of the various DNA bands in the gel is then recorded photographically.

4. Recovery of Dna from Agarose Gel Recovery of DNA from Agarose gels • DNA can be recovered from agarose gels thru different methods such as electro-election, freeze-squeeze, freeze-thaw, and also by low-melting–point agarose.

5. Recovery of Dna from Agarose Gel LMP – Agarose G- Recovery of DNA • Commercially available agarose is not 100% pure; it is contaminated with other polysaccharides, salts and proteins. The amount of contamination varies from batch to batch of agarose and affects both • the migration of the DNA • the ability of the DNA recovered from the gel to serve as substrate in enzymatic reactions.

6. Recovery of Dna from Agarose Gel • Because of the great increase in demand during the past years, manufacturers prepare special grades of agarose that are free of contaminants, e.g. inhibitors and nucleases. • There are also chemically modified forms of agarose that gel and melt at low temperatures without significant deterioration in the strength of the hardened gel.

7. Recovery of Dna from Agarose Gel • Introduction of the hydroxy-ethyl (O-C2H5) group into the polysaccharide chain causes the agarose to gel at approximately 30ºC and to melt at ~65ºC; this is well below the melting temperature of most dsDNA. • Because low melting temperature agarose remains still fluid at 37ºC, enzymatic manipulations, such as restriction enzyme digestions and ligations can be carried out by adding portions of the melted gel slice directly to the reaction mixture.

8. Recovery of Dna from Agarose Gel • The agarose decreases the efficiency of the reactions, even if it is pure, thus in these reactions the enzyme amount has to be increased 4-5 fold. • Therefore, in most cases, DNA is purified from the gel first and then used in subsequent steps. • The efficiency with which DNA is recovered from agarose gels is a function of its molecular weight. As the size of the DNA increases, the yield progressively decreases.

9. Recovery of Dna from Agarose Gel Procedure: • With a sharp scalpel, cut out the slice of agarose containing the DNA and transfer it to a sterile tube. • Add LMP-agarose buffer to the tube and incubate at 65ºC to melt the gel.

10. Recovery of Dna from Agarose Gel • Extract DNA 1x with Phenol and 2x with Chloroform. Recover the aqueous phase each time by centrifuging for 10 minutes at 5000rpm at RT. The white substance at the interphase is agarose. • Transfer the aqueousphase to a new tube, add 0,2 vol. of 9.5 M ammonium-acetate and 2 vol. of cold absolute EtOH; mix by inverting the tube.

11. Recovery of Dna from Agarose Gel • Wait for 10 minutes at room temperature; recover DNA by centrifugation at max speed for 15 minutes. • Dry the pellet and dissolve it in TE-buffer or water. • Chech the DNA on a 2% agarose gel, at 200 V for 8-10 minutes designed and prepared by alburse