Vector NTI使用介绍(PPT).ppt

* * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * Advanced Molecule Design with Complicated Recipient Step 5: Design and inspect the new molecule After the Design Preferences are set, click the Start Design button. Close the emptied Lists dialog box and go to the window that displays the newly designed Tutorial3 molecule. Inspect the new molecule and info in the Text Pane. * 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. 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 recommends oligos or PCR primers for clone analysis. 3. Print the Design Description: Open the Design Description folder Step#1 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 pre-determined 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 blunt-blunt 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 DonorStep 1: Define the recipient fragment