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)
☼ 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
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
☼ Translated from DNA molecules
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 selected
by users.
 Restriction sites are
analyzed and generated
by Vector NTI.
 Methods of terminus
modification are
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; ExtraChromosome 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.
2.
3.
4.
After the terminus is modified, click the Run button on the Lists dialog
box to launch the Construct Molecule dialog box.
Click the Construct button and select Tutorial as the subset, then click the
Overwrite button.
Close the emptied Lists dialog box and go to the window that displays the
newly constructed Tutorial1 molecule.
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.
2.
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.
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; ExtraChromosome 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.
2.
3.
4.
5.
6.
In the Design Parameter dialog box, click the Preference button
Choose your preferred parameters to create new molecules.
In this example: deselect Ligation-Blunt…Blunt option, so Vector NTI will
ensure all fragments have at least one cohesive end.
Leave other parameters at their default setting.
The Advanced Preferences allows you to change the way Vector NTI
evaluates possible design paths.
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.
2.
3.
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 Tutorial2 molecule.
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.
2.
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.
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
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).
4.
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.
2.
3.
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.
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.
2.
3.
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 Tutorial4 molecule.
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
new gel.
on the Main Toolbar to create a
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
3.
Molecules Subset: e-gel > Molecules: direct-clone; Source Enzymes Subset: MAIN
> Enzymes: Xmal
In the Sample Name box: enter Sample 1; in the Description box: enter directclone cut by Xmal. Click Add to Gel button.
Add the inverted-clone (cut by Xmal) to the same gel as Sample 2.
4.
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
Window Toolbar
2.
In the Choose Database Gel Marker dialog box, select SPP-EcoRI for
Lane 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.
3.
on the
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.
Click the Step Forward or Step Back button to see incremental
electrophoresis progress, or click the Animate button to view continuous
gel run.
3.
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.
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.
3.
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