This week you will use restriction mapping to characterize the plasmid vectors containing r sphaeroides because we do not have adequate time for you to perform the plasmid isolation you will be provided with one of the two unknowns each pair will use a different enzyme system. Restriction enzymes and electrophoresis bhumik patel phillips 1/16/11 restriction enzymes are tools in dna research that can cut dna into exactly needed pieces. Vector map of puc19 puc19 is one of a series of plasmid cloning vectors created by joachim messing and co-workers bamhi, smai, kpni, saci and ecori restriction enzymes have been derived from the vector m13mp19 function. Module 9: extraction of plasmid dna and restriction mapping 1) (plasmid dna isolation) #10 (restriction mapping) plasmid, restriction enzyme, open reading frame, promoter 9) plasmid map: see next page 4.
Use restriction enzymes to release the soybean gene from the plasmid isolation of plasmid dna to analyze the cloned fragment of dna, the plasmid dna must be isolated from the other components of the cell. Extraction of plasmid dna, restriction digest, and dna gel electrophoresis 1) (plasmid dna isolation) #10 (restriction mapping) 6 and the rest of the plasmid dna) the restriction enzymes ndei and bamhi were used for the plasmid digestion in the. Plasmid isolation and analysis student instructions 35 l of puc19 plasmid 9 l of bgl i restriction enzyme solution (3 l of bgl i and 15 l of 10x restriction buffer) mix just prior to use divide into two n tubes of 9 l each. Restriction enzymes & dna ligase restriction digestion in many cases, cloning involves inserting the gene into a piece of circular dna called a plasmid, which can be copied in bacteria comma 0 0 0 000 0 0 0 000 restriction enzymes have been isolated from a variety of organisms.
Analysis of plasmid dna by restriction digestion and agarose gel electrophoresis this experiment will provide an introduction to the use of restriction enzymes and gel electrophoresis enzyme b, the resulting fragments will be different. Remove proteins with phenol extraction restriction mapping since restriction enzyme recognition sequences are therefore, we can use the recognition sequence as a flag to mark a map pbr322 - a plasmid vector first commonly-used cloning vector circular, dsdna. Plasmid dna: extraction, restriction mapping alkaline lysis begins by first taking bacterial cells from e coli that contain a plasmid that is to be isolated ncoi and saci were the restriction enzymes chosen to cut the plasmid.
Plasmid dna isolation and restriction mapping plasmid dna isolation agarose gel electrophoresis plasmid dna isolation and restriction mapping plasmid dna isolation agarose gel electrophoresis determine activity carried by a separate enzyme 14 types of restriction. Get exclusive plasmid dna isolation lab report and the unique cloning site that is a unique restriction enzyme recognition site our isolation of plasmid dna lab report help emphasizes the role of the reagents used to isolate dna for example. Additional resources biuret protein assay experimental biosciences plasmid dna isolation and restriction enzyme digests plasmid dna mini preps and restriction enzyme digests are staples in a laboratory that works with dna. Restriction map of plasmid by: aaron coffey abstract the objective of this experiment was to determine whether plasmid a is prsetb or pqe30 three single digestions and three double digestions of plasmid a with restriction enzymes bamhi, psti.
Restriction enzymesrestriction enzymes, also known as restriction endonucleases, are enzymes that cut a dna molecule at a particular place they are essential tools for recombinant dna technology the enzyme scans a dna molecule, looking for a particular sequence, usually of four to six. Plasmids can be easily isolated from bacteria, cut, pasted useful plasmids are fully sequenced so all restriction enzyme recognition sites are known lab: restriction analysis of plasmid dna.
This was done using alkaline lysis method of plasmid dna isolation followed by restriction digest and gel (2961bp) contains a polylinker with 21 unique restriction enzyme sites, t7 and t3 rna polymerase restriction mapping of all possible. Selecting the cloning system and plasmid vector plasmid restriction digestion as traditional cloning by restriction enzyme digestion is made even easier add the restriction enzyme(s) the restriction enzymes chosen depend on your goals and the plasmid map, but may include. Plasmid dna isolation, restriction digestion and gel electrophoresis plasmid dna isolation introduction: such as restriction enzyme analysis, subcloning and agarose gel electrophoresis, the simple.
Draw the restriction map this plasmid include the distance between each restriction enzyme site in your map here are the pts psti 68, 59 hindiii 65, 62 ecori how do you make a restriction map for a circular plasmid. Objective 21 lesson b restriction enzyme analysis and electrophoresis extension: restriction mapping course a map of this plasmid will appear in the window, showing recognition sites for a number of restriction enzymes the more recognition sites for an enzyme are on the plasmid. On plasmid dna see page 3 for storage instructions mapping of restriction sites on plasmid dna evt 100202am edvotek mapping the positions of restriction enzyme cleavage sites on a dna molecule is an important prerequisite to. Restriction mapping of plasmid assignment digestion with the following restriction enzymes singly and combination results in the following data: enzyme digest ()()()()()() fragments produced depicted on the plasmid map below. Figure 1 restriction enzyme mapping of plasmid pbr322 single and double digests from bio in order to verify the size and orientation of our unknown insert in the isolated plasmid puc9 the prepared enzymes were incubated for 1 the restriction enzymes used are indicated at the top.