Rio | Restriction Enzyme

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  The procedure of this technique is divided into three steps:1.Digestion of total cellular DNA with one or more restriction enzymes and ligation of restriction half-site specific adaptors to all restriction fragments..!elective amplification of some of these fragments with two #$ primers that have corresponding adaptor and restriction site specific sequences.%.&lectrophoretic separation and amplicons on a gel matri'( followed )y visualisation of the )and pattern.The aim of this tool is to perform a theoretical A*+ - #$ e'periment )y using the same principles( and to suggest the adaptors and primers needed in the e'periment. Choosing   bacteria species   !elect the )acterial genera you want to wor, with. A form will appear where we may select the species( and the restriction enzymes and selective nucleotides for the e'periment. e may also include the plasmids if availa)le. *or )acterial species with two chromosomes( )oth chromosomes will )e used in the e'periment. n some  )acterial species( chromosomes are linear( and this fact has also )een considered in the e'periment. Digestion and ligation to adaptors   After selection of genome( the information necessary to perform the e'periment must )e selected. The form has )een partially reproduced )elow: /0-      -%0 !elect restriction enzymes from the lists  restriction enzyme 1  from $&1 list( and restriction enzyme 2  from $& list2. They will )e used to perform a complete theoretical DNA digestion of the genome. !elective nucleotides may also )e introduced see selective nucleotides in the form a)ove: 3A43 in the /0 end( and 34T3 in the %0 end2. The restriction enzymes availa)le in the form have the following characterictics:1.They all will cleave DNA within recognition sequence.The recognized sequence is not am)iguous no degenerated nucleotides2 %.They will all yeild overhang ends no )lund ends2   !elective nucleotides introduced in the form must match the upper strand of DNA see the grey segments in the picture2.As a result of DNA digestion( three types of DNA fragments will )e produced:  AG 3000 GT  1.*ragments cleaved in )oth ends )y the same restriction enzyme $&1 or $&2.*ragments cleaved in /0 end )y $&1 and )y $& in %0 end%.*ragments cleaved in %0 end )y $&1 and )y $& in /0 endn A*+ - #$( only type  and % fragments will )e amplified to yeild visi)le  )ands. This is due to ligation of different adaptors in each site of the fragment which will allow a geometric increase of this ,ind of )ands when #$ amplification is perform. As a consequence( type 1 fragments have )een eliminated from calculations in the theoretical e'periment. Adaptors will )e ligated to DNA fragments as shown in the picture )elow. +igation of adaptors in A*+ - #$ e'periments  The sequence of these adaptors are partially defined in the response page. !ome nucleotides of the adaptors are defined )y the recognition sequence of the restriction enzymes in pale )lue and pale green2. The sequence of adaptors which does not match the endonuclease recognition sequence in magenta and red2 must )e designed to avoid recognition of genomic DNA of the species used in the e'periment and to prevent a)errant results. f the restriction enzymes used to cut the genomic DNA are not heat la)ile( or restriction and ligation are performed simultaneously( the adaptor sequence must not regenerate the srcinal recognition sequence. To avoid this regeneration they must )e used adaptors must)e used that produce a )ase change in the recognition sequence. An e'ample is shown )elow: //-------GAATTC------//------TTAA------////-------CTTAAG------//------AATT------//  Eco RI Mse IDNA sequence with Eco RI and Mse I recognition sequences  //-------G AATTC------//------T TAA------////-------CTTAA G------//------AAT T------//DNA restrictionNNNNNNA AATTC------//------T TACnnnnnnnnnnnnTTTAA G------//------AAT GNNNNNNNNNNNNAAATTC------//------TTACnnnnnn nnnnnnTTTAAG------//------AATGNNNNNN Addition of adaptors. As nucleotides in red are different to the srcinal ones (lue! restriction sites are not reconstructed. n the results page of theoretical A*+ - #$ partial sequence of adaptors allow the reconstruction of   th srcinal recognition sequence( which is a valid option only when heat la)ile endonucleases are used( and restriction and ligation are  performed separately. PCR amplification with adaptor specific primers     n A*+ - #$ e'periments( the primers must )e designed to allow #$ amplification of the fragments cleaved )y $&1 in /0 end and $& in %0 end( so that they will )e complementary to sequence defined )y adaptors and sequence recognised )y restriction enzymes. The amplification will )e performed as shownin the picture:
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