MiniPrep and Restriction Digests

MiniPrep and Restriction Digests - BCHM464 Spring 2011...

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Mini-Prep & RestrictionDigest Analysis of AP Mutant Clones by Digestion with Restriction Endonucleases I. Introduction The purpose of this exercise is to isolate plasmid DNA from several transformants of the mutagenesis reaction and to determine whether any of the clones contain the desired mutation by screening for a change in the digestion pattern of a DNA restriction endonuclease. In order to isolate plasmid DNA from the transformed cells, small liquid cultures will be grown from individual colonies. There are several procedures available for purifying plasmid DNA in small quantities (commonly known as a plasmid "mini-prep"). After the cells are lysed, and the debris removed by centrifugation, you will use commercial QIAprep spin columns (silica-gel columns purchased from QIAGEN) to extract the plasmid DNA from the crude lysate. The separation is based on selective adsorption of plasmid DNA onto silica in high-salt buffer and elution in low-salt buffer. Although this procedure is more expensive than traditional protocols, it has the advantage of being easy, very rapid, and giving superior results. Restriction endonucleases are enzymes that produce double-stranded cuts in DNA at a particular recognition sequence. In general, they are highly specific for their target sequence, or restriction site. Enzymes that recognize 6 bases or more are the most useful, as their recognition sequences typically occur only a few times within a plasmid (3-20 kilobases). A large number of restriction enzymes are commercially available. These enzymes are capable of recognizing a variety of sequences. All of the amino acid changes used in this laboratory have been designed such that the nucleotide sequence of the mutant will create or destroy a restriction site in the plasmid. You will use a specific restriction enzyme to determine which of the transformants, if any, contain the desired mutation. Along with your mutagenic oligonucleotide, you should have received information indicating which enzyme to use for this purpose. The optimal conditions for each restriction endonuclease are slightly different. Before you begin the laboratory, you will be asked to look up what the recommended conditions are for the enzyme you will use. In addition, enzymes are often supplied in different concentrations. You will need to determine how many units and what volume of enzyme solution to use. From this information, you will develop a protocol for carrying out the digestion reactions. This protocol will be due before the start of lab and will be checked by the TA. After the plasmid DNA has been cleaved by the endonuclease, the size of the resulting DNA fragments can be determined by electrophoresis on a horizontal agarose gel. The DNA samples are added to one end of the gel, and an electric field is applied to the gel. The negatively charged DNA will move towards the positive electrode in the electric field. The agarose forms a stiff matrix that impedes the progress of the DNA molecules according to their size, so that small
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This note was uploaded on 03/01/2011 for the course BCHM 464 taught by Professor Staff during the Spring '08 term at Maryland.

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MiniPrep and Restriction Digests - BCHM464 Spring 2011...

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