【正文】 sign Molecule dialog box, click the Design button 2. Select the previously created Tutorial subset for the new molecule, click OK to continue. 3. In the Design Parameters dialog box, leave all settings at their default values. 4. Click the Preferences button, notice the bluntblunt ligation box remains turned off. 5. Leave everything at their default settings click OK to accept the Design Preference return to the Design Parameters dialog box. Advanced Molecule Design with Complicated Recipient Step 5: 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 Tutorial3 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: ? Recipient: partial digestion 1 ApaLI site inside recipient fragment. ? Donor: both termini were cut and then filled in, and ApaLI linkers were attached to the blunt ends before full digestion. ? Ligation: cohesive termini at both junctions. ? Enzymes to analyze and isolate insert with correct orientation: AvrII and ApaLI. ? Vector NTI also remends oligos or PCR primers for clone analysis. 3. Print the Design Description: Open the Design Description folder Step1 subfolder, click the Print Active Pane button on the tool bar. Advanced Molecule Design with Complicated Recipient Step 6: Inspect and print the design plan Advanced Molecule Design II – Complex Donor Fragment ? Task – Insert SV40’s LARGE_T gene from SV40 to a predetermined section of BPV1. ? Donor fragment: SV40 LARGE_T gene (5’ end with 440 bp flank region, 3’ end at NcoI site) ? Recipient fragment: BPV1 from 5000 to 2500 bp ? ligation: no bluntblunt ? Getting started 1. Open SV40 and BPV1 in Vector NTI 2. Arrange the display window to display the 2 molecules on the same screen: ? Select Menu Window Tile Vertical Advanced Molecule Design with Complex Donor Step 1: Define the recipient fragment 1. Activate the BPV1 Graphic Pane and click the Add Fragment to Goal List button to open the Fragment Wizard 2. 1st screen: select Design Recipient fragment click Next 3. 2nd screen: select Set to a Position and enter 5000 as the start for the 5’ of the new fragment, click Next 4. 3rd screen: select Set to a Position and enter 2500 as the start position of 3’ for the new fragment, then click Finish then Add to List button. Advanced Molecule Design with Complex Donor Step 1: Define the donor fragment 1. Activate the SV40 Graphic Pane and click Add Fragment to Goal List button to open the Fragment Wizard 2. 1st screen: select Design Donor fragment click Next 3. 2nd screen: Move the cursor to click/select the LARGE_T signal, click Next 4. 3rd screen: select Leave terminus Undefined, click Next 5. 4th screen: select Use flank region no larger than and enter 440 bps (the flank region can also be defined by dragging the mouse cursor in the Graphic Pane from the 5’), click Next 6. 5th screen: for the 3’ terminus, select Use specific site, then SHIFT + Click on the NcoI site at nucleotide 38. 7. Click the Finish then Add to List button. 1. From the tool bar, click the Open Goal List button 2. Make sure the recipient (BPV1 fragment) is listed first. 3. Click on the SV40 fragment, and then click the Edit button to open Fragment Editor dialog box. Notice that the left terminus has a flank region while the right is defined with a Ncol site, thus make the donor plicated that what used in the previous example (both ends were defined). 4. Check the Inverted box to change the direction of the donor fragment to match the recipient’s direction and then click the OK button. Advanced Molecule Design with Complex Donor Step 3: Inspect the Goal Molecule Definition List Advanced Molecule Design with Complex Donor Step 4: Describing the new molecule 1. Click the Run button, in the Design Molecule screen, enter name ―Tutorial4‖ 2. Click the Recipient39。 ExtraChromosome Replication: Bacteria。 Keyword: your last name) 4. Click Add and then OK to return to the Design Molecule dialog box. Advanced Molecule Design with Complex Donor Step 5: Prepare to design and set the preferences 1. In the Design Molecule screen, click the Design button 2. Select the previously created Tutorial subset for the new molecule, click OK to continue. 3. In the Design Parameters dialog box, leave all settings at their default values. 4. Click the Preferences button, notice the bluntblunt ligation box remains turned off. 5. Leave everything at their default settings and click the OK button to accept the Design Preference and return to the Design Parameters dialog box. Advanced Molecule Design with Complex Donor 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 Tutorial4 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 mole