vnti11 basics course
Post on 06-Dec-2014
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DESCRIPTION
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basics course
Introduction
Introduction
Database Explorer
Molecule Display
Molecule Editing
Restriction Enzymes
Primer Design and PCR analysis
Molecule construction (cloning)
Introduction
Download VectorNTI11 from Invitrogen
http://www.invitrogen.com/site/us/en/home/LINNEA-Online-Guides/
LINNEA-Communities/Vector-NTI-Community/Vector-NTI.html
After installation the software will run in DEMO mode
until it is unlocked by a static or dynamic license.
Dynamic licenses are available by VIB (BITS).
Overview modules
VectorNTI Explorer:
The database to store your data
VectorNTI:
Sequence creation, mapping, analysis,
design, annotation, illustration and
molecular biology data management
VectorNTI Explorer
VectorNTI Explorer
VectorNTI Explorer
New Object
VectorNTI Explorer
Organize your objects into groups using subsets
(and separators)
New Subset
VectorNTI Explorer
Organize your objects into groups using subsets
separator
Subset
VectorNTI Explorer
New Object
VectorNTI Explorer
New Object
VectorNTI Explorer
New Object
VectorNTI Explorer
New Object
VectorNTI Explorer
New Object
VectorNTI Explorer
VectorNTI Explorer
All molecules from all subsets are visible under MAIN
NEWMOL is in the MAIN set, not under any subset
VectorNTI Explorer
All molecules from all subsets are visible under MAIN
NEWMOL is in the MAIN set, not under any subset
Place NEWMOL in a subset by clicking, holding and moving the
name under MAIN to the subset name (DRAG and DROP)
VectorNTI Explorer
Subsets and separators can be moved in a similar way
DELETE = delete from database
DISMISS = remove subset (molecules NOT removed)
CLEAR – EXCLUDE = remove molecules from subset, NOT from MAIN database
VectorNTI Explorer
Subsets and separators can be moved in a similar way
DELETE = delete from database (= pressing ‘delete’!)
DISMISS = remove subset (molecules NOT removed)
CLEAR – EXCLUDE = remove molecules from subset, NOT from MAIN database
VectorNTI Explorer
Subsets and separators can be moved in a similar way
DELETE = delete from database
DISMISS = remove subset (molecules NOT removed)
CLEAR – EXCLUDE = remove molecules from subset, NOT from MAIN database
VectorNTI Explorer
subset selected
molecule selected
VectorNTI Explorer
VectorNTI Explorer
VectorNTI Explorer
We want to gather all NCBI molecules in a separate subset for easy access
VectorNTI Explorer
VectorNTI Explorer
VectorNTI Explorer
VectorNTI Explorer
VectorNTI Explorer
VectorNTI Explorer
VectorNTI Explorer
VectorNTI Explorer
VectorNTI Explorer
Backup on other location
e.g. network drive or external drive
Local database on network drive
» use same database on ≠ computers
We want to add some more structured information to each entry -> new field!
VectorNTI Explorer
New field Import fields
VectorNTI Explorer
VectorNTI Explorer
VectorNTI Explorer
VectorNTI Explorer
VectorNTI Explorer
VectorNTI Explorer
VectorNTI ExplorerPaste the data
in e.g. Excel
VectorNTI Explorer
Summary
» Create, edit and delete objects and subsets
» Organize objects into convenient groups (subsets)
» Import and export objects and archives
» Perform database searches
VectorNTI Explorer
Exercises
» Import restriction enzymes from REBASE
http://rebase.neb.com (bairoch.nnn file)
» Add a new DNA marker in the Gel Markers database
» Select the pUC plasmids and export them into an archive file
» Make 2 RE subsets:
(1) BamHI, EcoRI, EcoRV, HindIII, NcoI,
NdeI, PstI, PvuI, SmaI and XmaI
(2) 6bp non-ambiguous
VectorNTI Explorerhttp://Rebase.neb.com/rebase/rebase.html
VectorNTI Explorer
VectorNTI Explorer
Save file as...
» bairoch.nnnR
VectorNTI Explorer
Exercises
» Import restriction enzymes from REBASE
http://rebase.neb.com (bairoch.nnn file)
» Add a new DNA marker in the Gel Markers database
» Select the pUC plasmids and export them into an archive file
» Make 2 RE subsets:
(1) BamHI, EcoRI, EcoRV, HindIII, NcoI,
NdeI, PstI, PvuI, SmaI and XmaI
(2) 6bp non-ambiguous
VectorNTI
Start VectorNTI...
VectorNTI
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MAIN MENU
Links to the other modules of the Vector NTI suite
VectorNTI
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MAIN TOOLBARChangeable toolbar
Depending on
which pane is
active
VectorNTI
...TEXT WINDOW
VectorNTI
... GRAPHICS WINDOW
VectorNTI
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SEQUENCE WINDOW
VectorNTI
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Local
Database
VectorNTI
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Open the local
database
VectorNTI
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Click pACYC177 to open
VectorNTI
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Resize windows
for a better view
VectorNTI
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Copy complete
graphics
1
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but COPY (Ctrl+C)
& PASTE (Ctrl+V)
also works...
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Open the local
database
VectorNTI
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VectorNTI
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Molecule display setup
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How to edit your
molecule
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Link Panes
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VectorNTI: Molecule Editing
Close My pBR322
Formatting the graphics of a molecule:
» NCBI nucleotide database: NM_001025366
VectorNTI: Molecule Editing
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Retrieve directly from
NCBI into VectorNTI:
» Tools » Open » Retrieve...
NM_001025366
VectorNTI: Molecule Editing
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VectorNTI: Molecule Editing
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VectorNTI: Molecule Editing
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VectorNTI: Molecule Editing
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VectorNTI: Molecule Editing
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VectorNTI: Molecule Editing
...Ctrl + click
» ARRANGEMENT
VectorNTI: Molecule Editing
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Graphics Display Setup
>> all features will be
displayed in their specific way
VectorNTI: Molecule Editing
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Feature Display Setup
>> change the way this
feature is displayed
VectorNTI: Molecule Editing
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VectorNTI: Molecule Editing
...Picture
Editing Mode
VectorNTI: Molecule Editing
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VectorNTI: Molecule Editing
Molecule display setup:
» deselect restriction map to hide the RE sites
Graphics display setup: new style for CDS feature
» Shape 0 + More... (fill color + darker color for border)
Save the molecule in the database
VectorNTI: Molecule Editing
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Change
graphical
display
VectorNTI: Molecule Editing
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VectorNTI: Molecule Editing
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VectorNTI: Molecule Editing
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VectorNTI: Molecule Editing
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VectorNTI: Molecule Editing
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VectorNTI: Molecule Editing
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VectorNTI: Molecule Editing
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VectorNTI: Molecule Editing
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VectorNTI: Molecule Editing
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VectorNTI: Molecule Editing
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VectorNTI: Molecule Editing
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VectorNTI: Restriction enzymes
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VectorNTI: Restriction enzymes
Import enzymes
» From archive » DBINIT.EA4
» From REBASE » baroich.nnn
Restriction map setup
Restriction analysis and report
Restriction digestion and gel display
Open pUC19 from the database
VectorNTI: Restriction enzymes
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VectorNTI: Restriction enzymes
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VectorNTI: Restriction enzymes
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Primer design and PCR analysis
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Primer design and PCR analysis
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Primer design and PCR analysis
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Primer design and PCR analysis
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Select 2 enzymes (use Ctrl)
e.g. PvuI and EcoRV
Molecule construction (cloning)
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Molecule construction (cloning)
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Molecule construction (cloning)
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Restriction Analysis and Report
Molecule construction (cloning)
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Molecule construction (cloning)
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Molecule construction (cloning)
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Molecule construction (cloning)
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Molecule construction (cloning)
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Molecule construction (cloning)
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Molecule construction (cloning)
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Molecule construction (cloning)
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Select BamHI and PvuI
Molecule construction (cloning)
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Molecule construction (cloning)
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VectorNTI: Restriction enzymes
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VectorNTI: Restriction enzymes
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VectorNTI: Restriction enzymes
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VectorNTI: Restriction enzymes
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VectorNTI: Restriction enzymes
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VectorNTI: Restriction enzymes
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VectorNTI: Restriction enzymes
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Primer design and PCR analysis
Primer design for PCR on GST from plasmid pGEX-5G/LIC
GST: 258-935 bp | MCS: BamHI (951), Xma (956), EcoRI (961)
Molecule construction: GST in pET104-DEST (NdeI – XmaI)
Primer design and PCR analysis
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Primer design and PCR analysis
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Primer design and PCR analysis
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Primer design and PCR analysis
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Primer design and PCR analysis
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Primer design and PCR analysis
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Primer design and PCR analysis
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Primer design and PCR analysis
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Primer design and PCR analysis
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Primer design and PCR analysis
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Primer design and PCR analysis
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Primer design and PCR analysis
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Primer design and PCR analysis
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Primer design and PCR analysis
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Primer design and PCR analysis
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Primer design and PCR analysis
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Primer design and PCR analysis
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Primer design and PCR analysis
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Primer design and PCR analysis
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Primer design and PCR analysis
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Primer design and PCR analysis
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Primer design and PCR analysis
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Primer design and PCR analysis
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Primer design and PCR analysis
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Primer design and PCR analysis
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Primer design and PCR analysis
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Primer design and PCR analysis
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Molecule construction (cloning)
With the Fragment Wizard you can construct
a new DNA molecule
Define the needed fragments for the
Goal Molecule Definition list
Molecule construction:
cloning GST in pET104-DEST (NdeI – XmaI)
Primer design and PCR analysis
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Molecule construction (cloning)
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Molecule construction (cloning)
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Molecule construction (cloning)
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Molecule construction (cloning)
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Molecule construction (cloning)
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Molecule construction (cloning)
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Molecule construction (cloning)
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hold shift key and click in RE!
else Fragment wizard reverts to 5’!
Molecule construction (cloning)
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Molecule construction (cloning)
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Molecule construction (cloning)
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Molecule construction (cloning)
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Molecule construction (cloning)
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Molecule construction (cloning)
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Molecule construction (cloning)
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Molecule construction (cloning)
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Molecule construction (cloning)
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Molecule construction (cloning)
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Molecule construction (cloning)
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hold shift key and click in RE!
else Fragment wizard reverts to 5’!
Molecule construction (cloning)
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Molecule construction (cloning)
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Molecule construction (cloning)
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Molecule construction (cloning)
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Molecule construction (cloning)
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Molecule construction (cloning)
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Molecule construction (cloning)
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Molecule construction (cloning)
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Molecule construction (cloning)
Exercises
» Amplify the SV40 promoter from plasmid pRSVneo
Add a HindIII site to the 5’ end
and a SmaI site to the 3’ end
Save the result as SV40prom
» Clone the SV40 promoter using the
HindIII-SmaI in pUC18
Save as pUC18-SV40
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