【正文】 plorer Menu Table Import Molecules from Archive locate and open the Vector NTI workshop folder on the Desktop select click Open enter Subset Name ―egel‖ to import the tinsert and into the egel subfolder. 2. Open and arrange the display window to display the 2 molecules on the same screen. ? Construct the new molecule Identify the desired clones use Vector NTI simulated gel electrophoresis tool ? Question – In the previous exercise, the ligation conditions permit all possible donor orientations. How to identify the clones in which the tinsert is cloned in the desired direction? ? Answer – Perform restriction digestion analysis to analyze the clones. ? Vector NTI Solution – Before conduct restriction digestion analysis, use Vector NTI’s simulated gel electrophoresis to configure and predict digestion patterns for different clones. Use Vector NTI simulated gel electrophoresis to identify desired clones Step 1: Create a new gel with desired electrophoresis parameters 1. Click the New Gel button on the Main Toolbar to create a new gel. 2. In the Gel Setup dialog box, select Example of Agarose Gel from the list of Electrophoresis Profile. You may modify all the settings and create your favorite Electrophoresis Profile. Use Vector NTI simulated gel electrophoresis to identify desired clones Step 2: Create samples and add to the gel 1. In the Gel Display Window, click the Create Sample button on the Window Toolbar. 2. In the Create Gel Samples dialog box, make the following selection: Source Molecules Subset: egel Molecules: directclone。 Source Enzymes Subset: MAIN Enzymes: Xmal 3. In the Sample Name box: enter Sample 1。 in the Description box: enter directclone cut by Xmal. Click Add to Gel button. 4. Add the invertedclone (cut by Xmal) to the same gel as Sample 2. Use Vector NTI simulated gel electrophoresis to identify desired clones Step 3: Add Gel Marker to the gel 1. In the Gel Display Window, click the Add Marker Lane button on the Window Toolbar 2. In the Choose Database Gel Marker dialog box, select SPPEcoRI for Lane 3. 3. Create the Lamda HindIII marker: select Menu Gel Create Gel Marker in the New Gel Marker dialog box, enter the name. In the Gel Marker tab, enter the each fragment (23130, 9416, 6557, 4361, 2322, 2027 and 560) click OK. Add this marker to the Lane 4. Use Vector NTI simulated gel electrophoresis to identify desired clones Step 4: Run the Gel 1. In the Gel Pane, click TrueScale View button. You may also choose the Fit to Window button to maximize the gel display. 2. Enter 1:30 in the time indicator box and press the Enter key, the gel display is set for 1 h 30 min run. 3. Click the Step Forward or Step Back button to see incremental electrophoresis progress, or click the Animate button to view continuous gel run. Use Vector NTI simulated gel electrophoresis to identify desired clones Step 5: Inspect the Gel Display Window Text Pane 1. Notice for each sample, the number of fragments, and the properties of each fragment is listed. 2. Notice that the Source link for each fragment is also displayed, which directly lead users to the fragment in the Graphic Pane of the original molecule. 3. You can change the color for a fragment: select a fragment right click mouse and choose Sample Fragment Properties in the subsequent dialog box, choose desired color and line pattern. Use Vector NTI simulated gel electrophoresis to identify desired clones Step 6: Other Operations with Gel Display 1. Estimate Fragment Separation time: Use the mouse cursor to highlight/select the target fragments, and then click the Calculate Separation Time button. 2. Save the Gel Display Window: select Menu Gel Save as Gel Document enter a name and the gel is saved in gel document format. The document can be opened by selecting Menu Gel Open Document. 3. Copy Gel Display Window Data: select the desired pane then click the Camera button make further selections in the Camera dialog box before make the copy. 4. Print the Gel Display Window: activate the intended pane by mouse clicking it click the Print Active Pane button. ? Vector NTI Advance 10 offers an interlocking set of putational tools for planning Gateway or TOPO cloning experiments from beginning to end. ? It allows you to simulate rebinant strategy, as well as to validate design of your reagents before proceeding to primer ordering, benchtop operations, etc. Steps necessary to identify and screen transformants, expedite purification, fuse an insert with vector tags, etc. can be identified and modifications made before investing time and money. ? Vector NTI tools can be used to plan individual experiments (single or multisite cloning) or to design batch cloning experiments. In Silico Molecular Cloning: ComputerAided Experimental Guidance Vector NTI Advance? Software for Gateway174。 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