Vector NTI molecule construction design workshop by HC11112904329

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									Molecule Construction and Design
 Using Vector NTI 10 Advance


            Yi-Bu Chen, Ph.D.
         Bioinformatics Specialist
          Norris Medical Library
     University of Southern California


             323-442-3309
      yibuchen@belen.hsc.usc.edu
              Workshop Outline
A. Overview of molecule types and creation methods
B.   Major steps and tools for molecule construction and
     design
C. Construct a new DNA molecule
D. Design a new DNA molecule
E.   Overview of Gateway and TOPO cloning
F.   Using simulated gel electrophoresis to analyze
     cloned products
G. Useful online resources for molecule design purpose
     Fundamental Molecule Types in Vector NTI

 Basic Molecules (DNA/RNA/Protein)
  ☼ Notbuilt from component fragments
  ☼ Sequences and features are either entered by users or
  imported from other databases.
 Constructed DNA/RNA Molecules
  ☼ Built from one or more fragments (parent molecules,
  linkers, adapters, etc.)
  ☼ Automatically receive the Feature map and sequences from
  the parent molecules.
 Constructed Protein Molecules
  ☼ Translated from a coding sequence of a DNA molecule
  ☼ Does not receive the Feature map from its parent DNA
  molecule
  Methods of Creating New Molecule in Vector NTI

 Basic Molecules (DNA/RNA/Protein)
  ☼ Importing molecules or sequences
  ☼ Manually creating new molecules

 Constructed DNA/RNA Molecules
  ☼ Splicing   exons of an intron-exon join feature of another
  molecules
  ☼ Constructing from compatible component fragments from
  other molecules
  ☼ Designing from components of a user-defined fragments list
  ☼ Back-translating from protein molecules from components of
  a user-defined fragments list
 Constructed Protein Molecules
  ☼ Splicingexons of a gene
  ☼ Translated from DNA molecules
 Molecule Construction vs. Molecule Design

 Recipient and donor      Recipient and donor
  fragments are defined     fragments are selected
  by users.                 by users.
 Restriction sites are    Restriction sites are
  defined by users.         analyzed and generated
 When required, the        by Vector NTI.
  methods of terminus      Methods of terminus
  modification are          modification are
  defined by users.         determined by Vector
                            NTI.
     Major Tools for Molecular Construction and Design

 Fragment Wizard
  ☼ Construct fragments (define positions and termini)
  ☼ Design recipient/donor fragments (define termini)
  ☼ Add defined fragments to the Goal Molecule Definition List

 The Goal Molecule Definition List
  ☼ Contains  the list of fragments defined and added by the user
  using the Fragment Wizard
  ☼ Serves as the starting point of molecular construction and
  design
  ☼ Designs from components of a user-defined fragments list

 The Construct/Design Molecule Dialog Box
  ☼ Allows users to set the construction parameters and design
  preferences
  ☼ Allows users to enter information about the new molecule
     Major Steps for Molecule Construction

1. Use Fragment Wizard to define component
   fragments.
2. Add defined fragments to the Goal Molecule List.
3. Use the Construct Molecule Dialog Box to set
   construction parameters, including necessary
   terminus modifications.
4. Name, select data and describe the new molecule.
5. Verify and edit the component fragments in the Goal
   Molecule Definition List.
6. Initiate molecule construction.
       A Molecule Construction Example

 Task – Clone a fragment from pBR322 into pUC19
  ☼ Donor   fragment: pBR322, 5’EcoRI—3’AvaI
  ☼ Recipient   fragment: pUC19, 5’SmaI—3’EcoRI

 Getting started
  1. Open pBR322 and pUC19 in Vector NTI

  2. Arrange the display window to display 2 molecules on the
  same screen:

   ☺ Select Menu > Window > Tile Vertical
              A Molecule Construction Example
Step 1: Describe component fragments in the Fragment Wizard

A. Define the recipient pUC fragment (5’SmaI—3’EcoRI)
   1. Activate the pUC Graphic Pane
   2. Click Add Fragment to Goal List button to open the Fragment Wizard
   3. 1st screen: select Construct fragment > click Next
   4. 2nd screen: Click on the SmaI (415 bp) site in the graphic pane to set the
   5’ terminus. Click Next
   5. 3rd screen: hold the SHIFT key and click on the EcoRI site in the
   graphic pane to set the 3’ terminus. Click Finish
   6. Check the description of the fragment in the New Fragment message
   box. Click Cancel to go back to the Fragment Wizard if there are errors,
   otherwise, click Add to List to add the 1st fragment to the Molecule Goal
   List.
B. Define the donor pBR322 fragment (5’EcoRI—3’AvaI)
   following the above steps
               A Molecule Construction Example
                 Step 2: Inspect the Goal List

1. From the tool bar, click the Open Goal List button
2. Notice that the 1st fragment in the list is always considered as
   the ―recipient‖ molecule; the order of the fragments can be
   changed by click the Up or Down buttons
3. If no errors, click the Run button in the Lists screen
               A Molecule Construction Example
              Step 3: Describing the new molecule
1. In the Construct Molecule screen, enter name ―Tutorial1‖
2. Click the Recipient's Start button to define the start of the
   new molecule (can be set at any other positions)
3. Click General Info button to enter more info in the General
   Data dialog box (Description: Tutorial molecule#1; Extra-
   Chromosome Replication: Bacteria; Replicon Type: plasmid; Keyword:
   your last name)
4. Click Add and then OK button to return to the Construct
   Molecule dialog box.
               A Molecule Construction Example
      Step 4: Construct the new molecule/Initial attempt


1. Click the Construct button, in the Insert Molecule to Subset
   dialog box, enter ―Tutorial‖ as subset name, and then click
   OK button.
2. Vector NTI warns incompatible fragments (recipient pUC19
   fragment’s blunt 5’ SmaI terminus cannot be matched with
   the pBR322 cohesive 3’AvaI terminus).
3. Click the OK button to acknowledge the messages and return
   to the Construct Molecule dialog box > Click the Close button
   to return
4. You now need to modify one of the donor termini to make it
   compatible with the recipient termini for successful ligation.
                A Molecule Construction Example
     Step 4: Construct the new molecule/modify the terminus
1. To make the 2 fragments compatible, you need to modify the
   pBR322 cohesive 3’ AvaI terminus (5’overhang) into a blunt one.
2. In the Lists screen, click to select the pBR322, then click the Edit
   button.
3. In the Fragment of Molecule dialog box, click the Right
   Terminus button.
4. In the Right Terminus dialog Box, select Completely filled in in
   the Biochemical Operations section; then click OK.
               A Molecule Construction Example
 Step 4: Construct the new molecule/complete 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: Re-construct 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
     Re-construct from the short-cut menu.
2.   Make any desired changes in the Construct Molecule dialog box, and click
     the Construct button to re-construct the molecule.
           Major Steps for Molecule Design
1. Define the goal molecules
   ☼ Use the Fragment Wizard to define recipient and donor fragments
   ☼ Place the fragments in the Goal Molecule Definition List in proper order.

2. Inspect the Goal Molecule Definition List.
3. Enter information for the new molecule in the Design Molecule
   dialog box.
4. Set appropriate parameters and design preferences in the
   Design Parameter dialog box.
5. Start the designing process.
6. Inspect the design plan under the Design Description folder in
   the Text Pane.
7. If not satisfied, re-design the molecule by changing the goal
   molecule description or using different design parameters.
      A Simple Molecule Design Example

 Task – Clone a fragment from pBR322 into pUC19
  ☼ Donor  fragment: pBR322, the TC(R) signal
  ☼ Recipient fragment: pUC19, 5’ @ 500 bp, 3’ @ 250 bp


 Getting started
  1. Open pBR322 and pUC19 in Vector NTI

  2. Arrange the display window to display 2 molecules on the
  same screen:

   ☺ Select Menu > Window > Tile Vertical
                 A Molecule Design Example
       Step 1: Define the recipient and donor fragments

A. Define the recipient pUC fragment
   1. Activate the pUC 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: for the 5’ of the new fragment, select Set to a position, and
   enter 500, click Next
   4. 3rd screen: enter 250 in the Set to a Position box to define the 3’
   terminus, click Finish then Add to List button.
B. Define the donor pBR322 fragment
   1. Activate the pBR322 Graphic Pane and click the 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 TC(R) signal.
   4. Click Finish then Add to List button.
                      A Molecule Design Example
                      Step 2: Inspect the Goal List
1.   From the tool bar, click the Open Goal List button
2.   Notice the Design button is selected, confirming the Design Mode.
3.   Make sure the recipient (pUC fragment) is listed first.
4.   Notice that the exact positions of donor are not defined yet (NODEF), but
     it must contain the TC(R) signal.
5.   If no errors, click the Run button in the Lists screen
                  A Molecule Design Example
              Step 3: Describing the new molecule

1. In the Design Molecule screen, enter name ―Tutorial2‖
2. Click the Recipient'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 molecule#1; Extra-
   Chromosome Replication: Bacteria; Replicon Type: plasmid; 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/Non-Ambiguous 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 Ligation-Blunt…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.
                            A Molecule Design Example
                      Step 7: Inspect and print the design plan

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 compatible.
     ☼ 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 pre-selecting properly ligated molecule before transformation.
     ☼ The new restriction site (AfeI) in the recombinant molecule that does not exist in
     the recipient allows one to use restriction analysis of the clones.
     ☼ Vector NTI also recommends 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 —
     Step#1 subfolder, click the Print Active Pane button  on the tool bar.
                      A Molecule Design Example
                  Step 8: Re-design 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 Re-design
     from the short-cut menu.
2.   Make any desired changes in the Design Molecule dialog box, and click
     the Design button to re-design 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; 5’ ApaLI site
  must be retained
  ☼ 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 Complicated Recipient
     Step 1: Define the recipient and donor Fragments
A. Define the recipient BPV1 fragment
   1. Activate the BPV1 Graphic Pane and click Add Fragment to Goal List
   button to open the Fragment Wizard
   2. 1st screen: select Design Recipient fragment > click Next
   3. 2nd screen: for the 5’ of the new fragment, click on the label of ApaLI
   site #2 (7631) in the Graphic Pane, click Next
   4. 3rd screen: select Save Site, and then click Next
   5. 4th screen: to define the 3’, press SHIFT+ Click on the same ApaLI site,
   Click Finish then Add to List button.
B. Define the donor SV40 fragment
   1. Activate the SV40 Graphic Pane and click the 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.
   4. Click Finish then Add to List button.
     Advanced Molecule Design with Complicated Recipient
       Step 2: Inspect the Goal Molecule Definition List
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 the
     Fragment Editor dialog box
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. (when the
     Inverted box is not checked, the system will design it either way).
   Advanced Molecule Design with Complicated Recipient
           Step 3: Describing the new molecule

1. Click the Run button, in the Design Molecule screen,
   enter name ―Tutorial3‖
2. Click the Recipient'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
   molecule#3; Extra-Chromosome Replication: Bacteria;
   Replicon Type: plasmid; Keyword: your last name)
4. Click the Add and then OK button to return to the
   Design Molecule dialog box.
   Advanced Molecule Design with Complicated Recipient
      Step 4: Prepare to design and set the preferences

1. In the Design 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 blunt-blunt
   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.
           Advanced Molecule Design with Complicated Recipient
                 Step 6: Inspect and print the design plan
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 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 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 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.
      Advanced Molecule Design with Complex Donor
      Step 3: Inspect the Goal Molecule Definition List

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
   complicated 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 4: Describing the new molecule

1. Click the Run button, in the Design Molecule screen,
   enter name ―Tutorial4‖
2. Click the Recipient'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
   molecule#4; Extra-Chromosome Replication: Bacteria;
   Replicon Type: plasmid; 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 blunt-blunt
   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.
                Advanced Molecule Design with Complex Donor
                   Step 7: Inspect and print the design plan


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:   full digestion with BamHI and NcoI.
     ☼ Donor: inverted, full digestion with BamHI and NcoI.
     ☼ Ligation: cohesive termini at both junctions.
     ☼ Confirm the clone: restriction digestion with BbeI (3 sites in the BPV1 vector vs.
     2 sites in the Tutorial4 molecule).
     ☼ Enzymes to isolate the insert: BamHI and NcoI.
     ☼ Vector NTI also recommends oligos or PCR primers for clone analysis.


3.   Print the Design Description: With the Design Description folder —Step#1
     subfolder open, click the Print Active Pane button on the tool bar.
    Vector NTI Molecule Construct and Design
  Exercise – A simple molecule construction

 Task – Clone the t-insert into pcDNA3.1’s EcoR1
  site#1 use molecule construct Method.
 Getting started – Import the required molecules
  1. In Vector NTI Explorer > Menu > Table > Import >
  Molecules from Archive > locate and open the Vector NTI
  workshop folder on the Desktop > select e-gel.ma4 > click
  Open > enter Subset Name ―e-gel‖ to import the t-insert and
  pcDNA3.1 into the e-gel 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 t-insert 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: e-gel > Molecules: direct-clone; Source Enzymes Subset: MAIN
      > Enzymes: Xmal
3.    In the Sample Name box: enter Sample 1; in the Description box: enter direct-
      clone cut by Xmal. Click Add to Gel button.
4.    Add the inverted-clone (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 SPP-EcoRI 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 True-Scale 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.
    Use Vector NTI for Gateway and TOPO Cloning

 Vector NTI Advance 10 offers an interlocking set of computational tools
  for planning Gateway or TOPO cloning experiments from beginning to
  end.
 It allows you to simulate recombinant strategy, as well as to validate
  design of your reagents before proceeding to primer ordering, bench-top
  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
  multi-site cloning) or to design batch cloning experiments.

In Silico Molecular Cloning: Computer-Aided Experimental Guidance
http://www.invitrogen.com/content.cfm?pageid=10212

Vector NTI Advance™ Software for Gateway® Cloning

http://www.invitrogen.com/content.cfm?pageid=10071&cid=fl-VNTIGATEWAY
                     GATEWAY Cloning Overview
1. What is it?
    Gateway Cloning Technology is Invitrogen’s universal cloning system based on
    bacteriophage lambda-based site specific recombination 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 attB-containing Expression Clones
    that can be propagated and expressed in a range of host cells for a given
    experiment.
4. What are the key commends in Vector NTI for the Gateway Cloning?
    In the Vector NTI main application window:
   Entry Clone – Menu|Cloning > Gateway Cloning > Create an Entry Clone
   Expression Clone – Menu|Cloning > Gateway Cloning > Create an Expression
    Clone by LR
   Destination Clone – Menu|Cloning > Gateway Cloning > Create a Novel
    Destination Vector
                       TOPO Cloning Overview
1. What is it?
    Invitrogen’s TOPO Cloning Technology relies on DNA topoisomerase I, which
    functions as both a restriction enzyme and a ligase. It effectively replaces the uses
    of restriction enzymes and ligase.

2. What is it for?
   Efficient cloning of Taq-amplified PCR products (up to 10 kb).
   Efficient cloning of blunt-end fragments
   Directional cloned products for protein expression (via the Gateway or other
    systems) using a variety of TOPO adapted vectors
   High throughput PCR (using the Multiplex PCR design)
   High throughput cloning

4. What are the key commends in Vector NTI for the TOPO Cloning?
    In the Vector NTI main application window:
   Generate a clone by TOPO cloning – Menu|Cloning >TOPO Cloning > Launch
    TOPO Wizard
   Generate a clone by PCR and TOPO cloning – Menu|Cloning > TOPO Cloning >
    Amplify selection to use in TOPO reaction
http://www.biocompare.com/jump/119/Vectors-Search.html
Online Resource for Molecule Design: PlasmID




     http://plasmid.med.harvard.edu/PLASMID/
                                         PlasmID in OBRC




http://www.hsls.pitt.edu/guides/genetics/obrc/dna/motifs_regulatory_sites/sequence_motif_search_alignment_manipulation/URL1174663391/info
          PlasmID Collection Overview




http://plasmid.med.harvard.edu/PLASMID/collection_overview.jsp
        PlasmID Plasmid Request Page




http://plasmid.med.harvard.edu/PLASMID/OrderOverview.jsp
                                                            http://seq.yeastgenome.org/vectordb /




http://www.hsls.pitt.edu/guides/genetics/obrc/dna/motifs_regulatory_sites/sequence_analysis_gene_prediction/URL1099412460/info
http://www.mrw.interscience.wiley.com/emrw/9780471142720/home
http://www.cshprotocols.org/
http://biowww.net/browse-62.html
Have questions or comments about this
workshop? Please contact us:

Yi-Bu Chen, Ph.D.
Bioinformatics Specialist
Norris Medical Library
University of Southern California

323-442-3309
yibuchen@belen.hsc.usc.edu

								
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