【正文】 Cloning Use Vector NTI for Gateway and TOPO Cloning GATEWAY Cloning Overview 1. What is it? Gateway Cloning Technology is Invitrogen’s universal cloning system based on bacteriophage lambdabased site specific rebination system (attL x attR ? attB x attP). It effectively replaces the uses of restriction enzymes and ligase. 2. What is it for? ? Protein expression ? Transfer DNA segments between different vectors while maintaining orientation and reading frame 3. What are the key steps? ? Insert the target sequence into an Entry Clone ? Transfer the target sequence into a variety of attBcontaining Expression Clones that can be propagated and expressed in a range of host cells for a given experiment. 4. What are the key mends in Vector NTI for the Gateway Cloni。 Source Enzymes Subset: MAIN Enzymes: Xmal 3. In the Sample Name box: enter Sample 1。 Replicon Type: plasmid。s Start button to define the start of the new molecule as the start of recipient molecule. 3. Click General Info button to enter more info in the General Data dialog box (Description: Tutorial molecule4。 Replicon Type: plasmid。s Start button to define the start of the new molecule as the start of recipient molecule. 3. Click the General Info button to enter more info in the General Data dialog box (Description: Tutorial molecule3。 Keyword: your last name) 4. Click Add and then OK button to return to the Design Molecule dialog box. A Molecule Design Example Step 4: Prepare to design the new molecule 1. In the Design Molecule screen, click Design button 2. Select the previously created Tutorial subset for the new molecule, click OK to continue. 3. In the Design Parameters dialog box, you can choose your restriction enzyme subsets, the transformation systems you use, and other parameters. 4. In this example, select Palindromes/NonAmbiguous REN subset, and leave other parameters at their default value. A Molecule Design Example Step 5: Configure preferences for molecule design 1. In the Design Parameter dialog box, click the Preference button 2. Choose your preferred parameters to create new molecules. 3. In this example: deselect LigationBlunt…Blunt option, so Vector NTI will ensure all fragments have at least one cohesive end. 4. Leave other parameters at their default setting. 5. The Advanced Preferences allows you to change the way Vector NTI evaluates possible design paths. 6. Click OK to accept all Preferences and return to the Design Parameters. A Molecule Design Example Step 6: Design and inspect the new molecule 1. After the design preferences are set, click the Start Design button. 2. Close the emptied Lists dialog box and go to the window that displays the newly designed Tutorial2 molecule. 3. Inspect the new molecule and info in the Text Pane. 1. Verify the restriction enzymes used in the design process. Add them to the display by using the Molecular Display Setup dialog box — Restriction Map Setup. 2. Inspect Design Plan – In the Text Pane, open the Design Description Folder and subfolder Step 1. Highlights of the bench instruction for creating the new molecule: ? No biochemical operations needed to modify the termini as they are patible. ? The selected cloning option gives the required orientation of the cloned pBR322 fragment in the pUC19 recipient ? One of the recipient’s restriction sites (SmaI) is lost after ligation, this gives a mean for preselecting properly ligated molecule before transformation. ? The new restriction site (AfeI) in the rebinant molecule that does not exist in the recipient allows one to use restriction analysis of the clones. ? Vector NTI also remends oligos or PCR primers for clone analysis. ? Vector NTI also lists restriction sites that can be used to isolate the closed fragments. 3. Print the Design Description: Open the Design Description folder —Step1 subfolder, click the Print Active Pane button on the tool bar. A Molecule Design Example Step 7: Inspect and print the design plan A Molecule Design Example Step 8: Redesign the new molecule 1. With the Tutorial Molecule 2 open in the Vector NTI, select Menu File Molecule Operations Advanced DNA/RNA Design to open the Design Molecule dialog box, click Yes to overwrite the original task and start the new design. Alternatively, in the Vector NTI Explorer window select the intended molecule right click the mouse choose Redesign from the shortcut menu. 2. Make any desired changes in the Design Molecule dialog box, and click the Design button to redesign the molecule. Advanced Molecule Design I – Complicated Recipient ? Task – Insert SV40’s LARGE_T gene from SV40 to the 2nd ApaLI site of BPV1. ? Donor fragment: SV40 LARGE_T gene (no ApaLI site) ? Recipient fragment: BPV1 at 2nd ApaLI site。 ExtraChromosome Replication: Bacteria。 then click OK. A Molecule Construction Example Step 4: Construct the new molecule/plete the construction 1. After the terminus is modified, click the Run button on the Lists dialog box to launch the Construct Molecule dialog box. 2. Click the Construct button and select Tutorial as the subset, then click the Overwrite button. 3. Close the emptied Lists dialog box and go to the window that displays the newly constructed Tutorial1 molecule. 4. Inspect the new molecule and information in the Text Pane Component Fragments folder. A Molecule Construction Example Step 5: Reconstruct the new molecule if needed 1. With the Tutorial Molecule 1 open in the Vector NTI, select Menu File Molecule Operations Advanced DNA/RNA Construct to open the Construct Molecule dialog box. Alternatively, in the Vector NTI Explorer window select the intended molecule right click the mouse choose Reconstruct