Advanced Workshops on Vector NTI

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Advanced Workshops on Vector NTI Part II
Molecule Construction and Design Yi-Bu
Chen, Ph.D. Ansuman Chattopadhyay, Ph.D. Health
Sciences Library System Molecular Biology and
Genetics Information Service University of
Pittsburgh
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Outline of the Workshop

1. Overview of molecule types and creation methods
2. Major steps and tools for molecule construction
   and design
3. Construct a new DNA molecule
4. Design a new DNA molecule
5. Using simulated gel electrophoresis to analyze
   cloned products

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Fundamental Molecule Types in Vector NTI

• Basic Molecules (DNA/RNA/Protein)
• Not built 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

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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
• Splicing exons of a gene
• Translating from DNA molecules

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Molecule Construction vs. Molecule Design

• Recipient and donor fragments are defined by
  users.
• Restriction sites are defined by users.
• When required, the methods of terminus
  modification are defined by users.

• Recipient and donor fragments are defined by
  users.
• Restriction sites are analyzed and generated by
  Vector NTI.
• Methods of terminus modification are determined
  by Vector NTI.

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Major Tools for Molecular Construction and
Molecular 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

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Major Steps for Molecule Construction

• Use Fragment Wizard to define component
  fragments.
• Add defined fragments to the Goal Molecule List.
• Use the Construct Molecule Dialog Box to set
  construction parameters, including necessary
  terminus modifications.
• Name, select data and describe the new molecule.
• Verify and edit the component fragments in the
  Goal Molecule Definition List
• Initiate molecule construction.

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A Molecule Construction Example

• Task Clone a fragment from pBR322 into pUC19
• Donor fragment pBR322, 5EcoRI3AvaI
• Recipient fragment pUC19, 5SmaI3EcoRI
• 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 gt Window gt Tile Vertical

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A Molecule Construction ExampleStep 1
Describe component fragments in the Fragment
Wizard

• Define the recipient pUC fragment
  (5SmaI3EcoRI)
• 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 gt click
  Next
• 4. 2nd screen Click on the SmaI 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
  (5EcoRI3AvaI) following the above steps

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A Molecule Construction ExampleStep 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

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A Molecule Construction ExampleStep 3
Describing the new molecule

1. In the Construct Molecule screen, enter name
   Turorial1
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 molecule1 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.

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A Molecule Construction ExampleStep 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 (pUC
   fragment blunt 5SmaI terminus cannot be matched
   with the pBR322 cohesive 3AvaI terminus).
3. Click the OK button to acknowledge the messages
   and return to the Construct Molecule dialog box gt
   Click the Close button to return
4. You now need to modify the termini to make them
   compatible for the ligation.

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A Molecule Construction ExampleStep 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 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.

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A Molecule Construction ExampleStep 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 gt Component Fragments folder.

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A Molecule Construction ExampleStep 5
Re-construct the new molecule

1. With the Tutorial Molecule 1 open in the Vector
   NTI, select Menu gt File gt Molecule Operations gt
   Advanced DNA/RNA gt Construct to open the
   Construct Molecule dialog box. Alternatively, in
   the Vector NTI Explorer window gt select the
   intended molecule gt right click the mouse gt
   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.

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

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A Simple Molecule Design Example

• Task Clone a fragment from pBR322 into pUC19
• Donor fragment pBR322, the TC(R) signal
• Recipient fragment pUC19, 5 _at_ 500 bp, 3 _at_
  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 gt Window gt Tile Vertical

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A Molecule Design ExampleStep 1 Define the
recipient and donor fragments

• 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
  gt 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.
• 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 gt
  click Next
• 3. 2nd screen Move the cursor to click/select
  the TC(R) signal.
• 4. Click Finish then Add to List button.

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A Molecule Design ExampleStep 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

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A Molecule Design ExampleStep 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 molecule1 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.

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A Molecule Design ExampleStep 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.

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A Molecule Design ExampleStep 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-BluntBlunt
   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.

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A Molecule Design ExampleStep 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.

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A Molecule Design ExampleStep 7 Inspect and
print the design plan

• 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
• 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 recipients restriction sites is
  lost after ligation, this gives a mean for
  pre-selecting properly ligated molecule before
  transformation.
• The new restriction site 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 Step1 subfolder,
  click the Print Active Pane button on the
  tool bar.

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A Molecule Design ExampleStep 8 Re-design the
new molecule

1. With the Tutorial Molecule 2 open in the Vector
   NTI, select Menu gt File gt Molecule Operations gt
   Advanced DNA/RNA gt 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
   gt select the intended molecule gt right click the
   mouse gt 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.

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Advanced Molecule Design I Complicated
Recipient

• Task Insert SV40s 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 gt Window gt Tile Vertical

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Advanced Molecule Design with Complicated
RecipientStep 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 gt 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 gt click
Next 3. 2nd screen Move the cursor to
click/select the LARGE_T signal. 4. Click
Finish then Add to List button.
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Advanced Molecule Design with Complicated
RecipientStep 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 recipients
   direction and then click the OK button. (when
   the Inverted box is not checked, the system will
   design it either way).

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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 molecule3 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.

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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 gt
   click OK to accept the Design Preference gt return
   to the Design Parameters dialog box.

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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.

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Advanced Molecule Design with Complicated
Recipient Step 6 Inspect and print the design
plan

• Verify the restriction enzymes used in the design
  process. Add them to the display by using the
  Molecular Display Setup dialog box gt 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 gtgt Step1 subfolder,
  click the Print Active Pane button on the tool
  bar.

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Advanced Molecule Design II Complex Donor
Fragment

• Task Insert SV40s 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 gt Window gt Tile Vertical

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Advanced Molecule Design with Complex DonorStep
1 Define the recipient fragment

• Activate the BPV1 Graphic Pane and click the Add
  Fragment to Goal List button to open the Fragment
  Wizard
• 1st screen select Design Recipient fragment gt
  click Next
• 2nd screen select Set to a Position and enter
  5000 as the start for the 5 of the new fragment,
  click Next
• 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.

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Advanced Molecule Design with Complex DonorStep
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 gt 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.

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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 recipients
   direction and then click the OK button.

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Advanced Molecule Design with Complex DonorStep
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 molecule4 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.

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Advanced Molecule Design with Complex DonorStep
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.

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Advanced Molecule Design with Complex DonorStep
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.

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Advanced Molecule Design with Complex DonorStep
7 Inspect and print the design plan

• 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 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 Step1 subfolder open,
  click the Print Active Pane button on the tool
  bar.

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Vector NTI Molecule Construct and DesignExercise
A simple molecule construction

• Task Clone the t-insert into pcDNA3.1s EcoR1
  site1 use molecule construct Method.
• Getting started Import the required molecules
• 1. In Vector NTI Explorer gt Menu gt Table gt
  Import gt Molecules from Archive gt locate and open
  the Vector NTI workshop folder on the Desktop gt
  select e-gel.ma4 gt click Open gt 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

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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 NTIs simulated
  gel electrophoresis to configure and predict
  digestion patterns for different clones.

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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.

44
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 gt Molecules direct-clone Source Enzymes
   Subset MAIN gt 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.

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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 gt
   Gel gt Create Gel Marker gt 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) gt click OK. Add this
   marker to the Lane 4.

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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 130 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.

47
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 gt right click mouse and choose Sample
   Fragment Properties gt in the subsequent dialog
   box, choose desired color and line pattern.

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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 gt Gel gt
   Save as Gel Document gt enter a name and the gel
   is saved in gel document format. The document
   can be opened by selecting Menu gt Gel gt Open
   Document.
3. Copy Gel Display Window Data select the desired
   pane gt then click the Camera button gt 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 gt click the
   Print Active Pane button.

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