To study the desired functionality of an insert there is a requirement of transferring them from one vector to another, this procedure of transferring is known as subcloning.\u00a0 Steps of the Subcloning:<\/p>\n
For any work on subcloning some prerequisite knowledge about the vector and insert is necessary.\u00a0 The information about the RE sites available in the parent vector multiple cloning regions, RE sites available the insert (in case of inset digestion). Also information about the recurrence of RE sites in destination vector multiple cloning regions and RE sites in the insert. Depending on the different combination of these sites, the subcloning strategy to be used is decided.<\/p>\n
A straightforward subcloning process can be utilized if the parent and destination vector multiple cloning regions contain common restriction sites and neither of these restriction sites occurs within your insert. Both the parent and destination vectors are digested using same two enzymes. This is followed by dephosphorylation of the destination vector. Separate both the insert and the dephosphorylated vector using an agarose gel. Then purify insert vector using any DNA purification system.\u00a0 Finally, these vectors and inserts are ligated.<\/p>\n
\u00a0<\/strong>There are cases when restriction sites in the parent and destination vector multiple cloning regions are not common but may be compatible. Compatible restriction sites have the same overhang sequence and can be ligated together. After ligation, the regenerated sites does not resemble both restriction sites in the parent and destination vector multiple cloning regions. Once the necessary enzymes for the ligation and restriction are identified. This cloning strategy also becomes straightforward.<\/p>\n The combination of common restriction sites leads to a possibility to only one match on one side of your insert. The issue should be addressed on the other side of the insert. A cut-blunt-cut can be used.\u00a0 The action of T4<\/span> DNA Polymerase can blunt any restriction site.\u00a0 Digest the parent vector and blunt that site with T4<\/span> DNA Polymerase. Run the products on a gel, purify and proceed with the common or compatible end restriction enzyme<\/span> digestion. Most vectors have at least one blunt-ended restriction site that can accept the newly created blunt end from the insert. If you don\u2019t have such a site or the site would not be in the correct orientation, the same \u201ccut-blunt-cut\u201d strategy may be applied to the destination.<\/p>\n \u00a0<\/strong>The final scenario of no common restriction site is common or compatible with the parent or destination vector.\u00a0 IN this scenario the best strategy is to amplify the insert with restriction sites in the primers to provide the compatibility. But this method has an own set of problems. There is a possibility of mutations.\u00a0 Also, there is a lot of difficulty in the digestion of the PCR<\/span> products. One more strategy can be used in this scenario. Cut out the insert with any enzymes. Treat with T4<\/span> DNA Polymerase to blunt either 5\u2032 or 3\u2032 overhangs and ligate into the destination vector opened with a blunt-end cutter or made blunt by T4<\/span> DNA Polymerase. Orientation of the isert may not be retained by this method.<\/p>\n Restriction endonucleases (RE), also referred to as restriction enzymes<\/span>, are proteins<\/span> that recognize short, specific (often palindromic) DNA sequences. Type II REs cleave double-stranded DNA (dsDNA) at specific sites within or adjacent to their recognition sequences. Many restriction enzymes<\/span> will not cut DNA that is methylated on one or both strands of the recognition site, although some require substrate<\/span> methylation. Restriction digestion is one of the most common reactions performed in molecular biology<\/span>.<\/p>\n Nuclease-Free Water \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 14\u03bcl<\/p>\n 10X Restriction Buffer \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 2\u03bcl<\/p>\n Acetylated BSA (1mg\/ml) \u00a0 2\u03bcl<\/p>\n DNA (~1\u03bcg) \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 1\u03bcl<\/p>\n Restriction Enzyme<\/span> (10u) \u00a0 1\u03bcl<\/p>\n Final Volume \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 20\u03bcl<\/p>\n Mix by pipetting and collect the contents at the bottom of the tube. Incubate at the appropriate temperature for the enzyme<\/span> for 1\u20134 hours. Add 4\u03bcl of 6X Blue\/Orange Loading Dye and analyze digested DNA by gel electrophoresis.<\/p>\n The presence of a restriction recognition site in the insert and the multiple cloning region does not necessarily preclude use of that restriction site in a subcloning strategy. Under normal restriction digest conditions, the enzyme<\/span> is in excess so that all recognition sites in the plasmid can be cleaved. You can manipulate the restriction digest conditions such that you will digest only a subset of sites. Many strategies have been employed to do partial digests: Decreasing reaction temperature, using a non-optimal buffer, and decreasing units of enzyme<\/span>. The method presented here uses dilutions of enzyme<\/span> in the optimal buffer<\/p>\n Preventing vector self-ligation is critical for reducing subcloning background. The efficiency of ligating the plasmid to itself is far better than ligating a separate piece of DNA into the vector and is the favored reaction. Removing the 5′ phosphates of the linearized vector will prevent T4<\/span> DNA Ligase<\/span> from recircularizing the vector. Calf Intestinal Alkaline Phosphatase is the classic enzyme<\/span> for vector dephosphorylation. The enzyme<\/span> can be used on 5′ recessed ends (i.e., results from an enzyme<\/span> leaving a 3′ overhang), 5′ overhangs and bluntends. After dephosphorylation, the enzyme<\/span> must be removed either by direct purification or gel electrophoresis and gel isolation with DNA purification systems like the Gel and PCR<\/span> Clean-Up System. Shrimp Alkaline Phosphatase can be used in place of Calf Intestinal Alkaline Phosphatase and offers the advantage of simple heat denaturation to inactivate the enzyme<\/span> without the need for further purification.<\/p>\n Dephosphorylating Vectors: Shrimp Alkaline Phosphatase Streamlined Restriction Digestion, Dephosphorylation and Ligation Procedure<\/p>\n Purification of the insert and destination vector are absolutely critical for success in subcloning applications. Years ago, each step called for phenol:chloroform extractions followed by ethanol precipitation to remove enzymes such as calf intestinal alkaline phosphatase from enzymatic vector manipulations. Guanidine-based nucleic acid<\/span> clean-up systems greatly simplified the removal of enzymes. Gel isolation methods further improved the efficiency of subcloning by segregating the wanted reactants from the unwanted reactants.<\/p>\n \u00a0<\/em><\/strong>Gel and PCR<\/span> Clean-Up System is designed to extract and purify DNA fragments directly from PCR<\/span>(a) or from agarose gels. Fragments of 100bp to 10kb can be recovered from standard or low-melt agarose gels in either Tris acetate (TAE) buffer or Tris borate buffer (TBE). Up to 95% recovery is achieved, depending upon the DNA fragment size. This membrane-based system, which can bind up to 40\u03bcg of DNA, allows recovery of isolated DNA fragments or PCR<\/span> products in as little as 15 minutes, depending on the number of samples processed and the protocol used. Samples can be eluted in as little as 15\u03bcl of nucleasefree water. The purified DNA can be used for automated fluorescent sequencing, cloning, labeling, restriction enzyme<\/span> digestion or in vitro transcription<\/span>\/ translation without further manipulation.<\/p>\n Agarose Gel Electrophoresis of DNA<\/p>\n Running double-stranded, linear DNA (like plasmid DNA from restriction enzyme<\/span> digests) on an agarose gel is a routine activity in molecular biology<\/span> laboratories. The basic method is very straightforward:<\/p>\n 1\u20135 volts\/cm. Higher voltages and shorter runs will decrease the resolution of the gel and may also cause overheating that may melt the agarose.<\/p>\n 10mM \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Tris-HCl, pH<\/span> 7.5<\/p>\n 50mM\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 EDTA<\/p>\n 15%\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Ficoll\u00ae 400<\/p>\n 0.03%\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 bromophenol blue<\/p>\n 0.03%\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 xylene cyanol FF<\/p>\n 0.4%\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 orange G<\/p>\n One or more dyes can be left out of the recipe to create a custom loading dye.<\/p>\n Dissolve 242g Tris base and 37.2g disodium EDTA, dihydrate in 900ml of deionized water<\/span>. Add 57.1ml glacial acetic acid and adjust the final volume with water to 1 liter. Store at room temperature or 4\u00b0C.<\/p>\n Dissolve 108g of Tris base and 55g boric acid in 900ml deionized water<\/span>. Add 40ml 0.5M EDTA (pH<\/span> 8.0) and increase the final volume to 1L. Store at room temperature or 4\u00b0C.<\/p>\n \u00a0<\/strong>DNA markers should always be run on agarose gels to aid in identifying bands of interest. This is especially true if you are performing applications such as partial restriction digestion. Promega offers a wide variety of DNA markers to fit your needs. BenchTop Markers come premixed with Blue\/Orange Loading Dye ready to load onto the gel. As the name implies, you can store them on your benchtop, no need to freeze and thaw every time you need it. Conventional markers are pure DNA solutions and come with a tube of 6X Blue\/Orange Loading Dye for use with the marker and your samples.<\/p>\n Molecular biologists have exploited DNA ligases to insert pieces of DNA into vectors for decades. The enzyme<\/span> most commonly used is derived from bacteriophage T4<\/span>. T4<\/span> DNA Ligase<\/span> is about 400-fold more active than E. coli<\/span> DNA ligase<\/span> for ligating blunt ends, and thus is the enzyme<\/span> of choice for all molecular biology<\/span> requirements. Promega offers T4<\/span> DNA Ligase<\/span> in standard or high-concentrate form, with the standard Ligase Buffer or with the 2X Rapid Ligation Buffer offered in the Rapid DNA Ligation System. The System allows rapid, 5-minute ligations for 5′ or 3′ overhang cohesive ends or 15-minute ligations for blunt ends.<\/p>\n \u00a0<\/strong>Rapid DNA Ligation System<\/p>\n Starting with a 1:2 molar ratio of vector:insert DNA when cloning a fragment into a plasmid vector is recommended.<\/p>\n The following ligation reaction of a 3kb vector and a 0.5kb insert DNA uses the 1:2 vector:insert ratio. Typical ligation reactions use 100\u2013200ng of vector DNA.<\/p>\n vector DNA \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 100ng<\/p>\n insert DNA \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 33ng<\/p>\n 2X Rapid Ligation Buffer \u00a0\u00a0 5\u03bcl<\/p>\n T4<\/span> DNA Ligase<\/span> (3u\/\u03bcl) \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 1\u03bcl<\/p>\n nuclease-free water to \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 10\u03bcl<\/p>\n \u00a0<\/span><\/strong>T4 DNA Ligase<\/span><\/p>\n Using a 1:1, 1:3 or 3:1 molar ratio of vector:insert DNA when cloning a fragment into a plasmid vector is recommended.<\/p>\n The following ligation reaction of a 3.0kb vector and a 0.5kb insert DNA uses the 1:3 vector:insert ratio. Typical ligation reactions use 100\u2013200ng of vector DNA.<\/p>\n vector DNA \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 100ng<\/p>\n insert DNA \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 50ng<\/p>\n Ligase 10X Buffer \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 1\u03bcl<\/p>\n T4<\/span> DNA Ligase<\/span> (3u\/\u03bcl) \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 1\u03bcl<\/p>\n Nuclease-Free Water to \u00a0\u00a0\u00a0 10\u03bcl<\/p>\n 22\u201325\u00b0C \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 3 hours \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Cohesive ends<\/p>\n 4\u00b0C \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Overnight \u00a0\u00a0\u00a0\u00a0\u00a0 Cohesive ends<\/p>\n 15\u00b0C \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 4\u201318 hours \u00a0\u00a0 Blunt ends<\/p>\n Controls help ensure that everything is functioning normally in your subcloning reaction. If something does go wrong, you can use your controls to figure out where a problem might have occurred. When ligating insert and vector, you can do a control ligation of vector with no insert. Carry this reaction through transformation and plating. The number of colonies you see can be a good indicator of how a ligation reaction performed and how many background colonies you will have on your plate.<\/p>\n <\/p>\n <\/p>\n <\/p>\n","protected":false},"excerpt":{"rendered":" To study the desired functionality of an insert there is a requirement of transferring them from one vector to another, this procedure of transferring is known as subcloning.\u00a0 Steps of the Subcloning: Release and purify your insert from the parent vector Ligate this insert into a prepared destination vector Transform this ligation reaction into competent […]<\/p>\n","protected":false},"author":4,"featured_media":0,"parent":401,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"","meta":{"_et_pb_use_builder":"","_et_pb_old_content":"","_et_gb_content_width":"","footnotes":""},"class_list":["post-1823","page","type-page","status-publish","hentry"],"_links":{"self":[{"href":"https:\/\/www.mybiosource.com\/learn\/wp-json\/wp\/v2\/pages\/1823","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.mybiosource.com\/learn\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/www.mybiosource.com\/learn\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/www.mybiosource.com\/learn\/wp-json\/wp\/v2\/users\/4"}],"replies":[{"embeddable":true,"href":"https:\/\/www.mybiosource.com\/learn\/wp-json\/wp\/v2\/comments?post=1823"}],"version-history":[{"count":0,"href":"https:\/\/www.mybiosource.com\/learn\/wp-json\/wp\/v2\/pages\/1823\/revisions"}],"up":[{"embeddable":true,"href":"https:\/\/www.mybiosource.com\/learn\/wp-json\/wp\/v2\/pages\/401"}],"wp:attachment":[{"href":"https:\/\/www.mybiosource.com\/learn\/wp-json\/wp\/v2\/media?parent=1823"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}Subcloning Strategy: Moving Inserts with Only One Common Site<\/strong><\/h3>\n
Subcloning Strategy: Blunt-End Method<\/strong><\/h3>\n
Methods<\/strong><\/h3>\n
\u00a0<\/strong>Restriction Digestion <\/strong><\/h4>\n
\n
\n
Partial Restriction Digestion<\/strong><\/h3>\n
Controlling Cut Frequency in Restriction Digestion <\/strong><\/h4>\n
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Dephosphorylating Vectors to Limit Self-Ligation<\/strong><\/h4>\n
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Purifying Vector and Insert<\/strong><\/h3>\n
Purifying Vector and Insert<\/strong><\/h3>\n
\u00a0<\/strong>Gel and PCR Clean-Up System<\/em><\/strong><\/h4>\n
\u00a0<\/em><\/strong>Gel Electrophoresis<\/em><\/strong><\/h4>\n
\n
\n
\n
Materials<\/strong><\/h3>\n
\n
\n
\n
DNA Markers<\/strong><\/h4>\n
Ligation: Ligating Vector and Insert<\/strong><\/h4>\n
Ligation<\/strong><\/h3>\n
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Ligation<\/strong><\/h3>\n
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Ligation: Control Reaction<\/strong><\/h3>\n