combined ape lectures fall 2021 - jorgensen.biology.utah.edu
TRANSCRIPT
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ApE Window
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ApE WindowNew window
Shift: Duplicate selection Open File
Save window Shift: Save as
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ShortcutsCommand Function
O Open
N New window
D Duplicate selected region
Shift-D Duplicate whole sequence
S Save sequence
Shift-S Save sequence as…
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ApE WindowSequence length
Edit lock
Cursor location Circular/ Linear
Dam/Dcm status
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ApE WindowSelection start
double click to setSelection length Selection end
double click to set
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ApE Window
%GC
ORF state Selection melting temperature
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Selection translation
The translation of the selected
region is shown here
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Selection translation
Right click here to set the
translation direction
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Preferences
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ApE WindowCut
Shift: Cut rev-comCopy
Shift: Copy rev-com
Paste Shift: Paste rev com
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ShortcutsCommand Function
X Cut
Shift-X Cut rev-com
C Copy
Shift-C Copy rev-com
V Paste
Shift-V Paste rev-com
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Copy Special
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ApE WindowFind
Shift: Clear find highlight
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ShortcutsCommand Function
F Find
Shift-F Clear find highlight
G Do find again
Shift-G New feature from find highlight
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Find dialog
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Find dialogYou can search for multiple patterns with a comma or
bar
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ApE Window
Open this to show the file
comment
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ShortcutsCommand Function
. New Feature
K Add features using feature library
Shift-K Clear all features
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EnzymesSelect Enzymes
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Select an enzyme to highlight
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Enzyme highlights
Enzymes can be highlighted
Enzyme name and sequence shows up here
when you put the mouse over the
sequence
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X-ray window
spacebar turns on the X-ray
window
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X-ray window
The X-ray window shows all the features
and enzyme sites in a row
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Side maps
First map shows the features in
the current lines
Second map shows the
features in the whole sequence
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Side maps
Click on any of these to select a
feature in the feature table and set the selection
to that feature
Side map features under the pointer are
listed here
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Virtual GelSelect Enzymes
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Virtual Gel
Click here to digest the sequence with each enzyme individually
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Virtual Gel
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Virtual Gel
Click here to digest the sequence with each enzyme at one time
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Virtual Gel
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Virtual Gel
Mouse here to highlight the band Double-click to select that region
in the sequence
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Virtual Gel
Mass % shows how much of the total DNA each band represents
Use this to decide how much DNA to load on a gel. 50 ng is about the
least DNA you can see on a gel.
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Virtual Gel
Click here to see a list of digest lanes
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Virtual Gel
Click and drag lanes to rearrange
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ShortcutsCommand Function
R Digest with currently selected enzymes
Shift-R Digest Dialog
E Enzyme selection dialog
Shift-E Set all functions to apply to selection
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Digestion dialog
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Digestion dialog
Each gel lane is a table row
Add and remove rows with these buttons
Rows can be dragged and duplicated
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Digestion dialog
Enzymes for digestions are added here
Add enzyme columns with this button
Show partial digest options with this
button
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Digestion dialog
Change what is in the lane with this menu
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Digestion dialog
Change the enzyme with this menu
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Digestion Dialog
Select the DNA or ladder for each lane
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Digestion DialogSelect EcoRI and SpeI
Add an Enzyme Column
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Digestion Dialog
You can also just create a set of new lanes with each open DNA
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Digestion Dialog
For convenience, you can digest a single DNA with all current
enzymes
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Digestion DialogPress this to do partial digests
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Digestion DialogPress this to select EcoRI in all
columns
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Digestion Dialog
This will digest pMLS280 and gfasPurple with EcoRI and SpeI
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Digestion Dialog
This will digest pMLS280 and gfasPurple with EcoRI and SpeI
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Restriction-Ligation Assembler
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Restriction-Ligation Assembler
Click and drag a gel lane here
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Restriction-Ligation Assembler
Click and drag a gel lane here
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Restriction-Ligation Assembler
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Restriction-Ligation Assembler
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Restriction-Ligation Assembler
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Restriction-Ligation Assembler
mouse here to see the feature names
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Restriction-Ligation Assembler
click here to flip a fragment
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Restriction-Ligation Assembler
click here to delete a fragment
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Restriction-Ligation Assembler
When everything works, you can click OK
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Restriction-Ligation Assembler
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Restriction-Ligation Assembler
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Feature library
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New Feature
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FeaturesCurrent features under the mouse pointer are listed
here
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Click this to hide/show the feature
table
Features
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Right-click here to edit a feature
Features
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Click this to sort the feature table
Features
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Command-click here to select
multiple features
Shift-click here to select a range
of features
Features
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Right-click here to edit a range of
features
Features
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Right-click here to edit a feature’s
direction
Features
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Right-click here to edit a feature’s type, or to raise, lower or hide all
of a type of feature
Features
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Right-click here to edit a feature’s
range
Features
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Shift-click this to hide/show the feature table
buttons
Features
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Golden Gate Assembler
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Golden Gate Assembler
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Golden Gate Assembler
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Golden Gate Assembler
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Golden Gate Assembler
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Golden Gate Assembler
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Golden Gate Assembler
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Golden Gate Assembler
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Golden Gate Designer
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Golden Gate Designer
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Golden Gate Designer
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Golden Gate Designer
You can’t use a fragment that has a
site already in it
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Golden Gate Designer
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Golden Gate Designer
Add fragments using this button
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Golden Gate Designer
Select these ranges
Then click “Next”
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Golden Gate DesignerCalculates the total
efficiency (on-target to off target)
Each fragment and the primers needed
You can set default values here bases
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Golden Gate Designer
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Golden Gate Designer
Re-start the calculation if it’s stuck
in an unfavorable search
Do a longer calculation from the current search position
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Golden Gate Designer
Generate the new product
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Golden Gate Designer
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Golden Gate Designer
The primers and PCR conditions are in the
comments
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Golden Gate Designer
The primers are new features in the feature
table
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Golden Gate Designer
The primers are new features in the
sequence
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Text map dialog
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Text map dialog
Each box is an analysis line
Turn off the line by unchecking this box
Drag the frames to change the order
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Text map dialog
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ORF map dialog
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ORF map dialog
Three forward frames
Three reverse frames
Longer than this are highlighted in color, and can be clicked
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Silent/ diagnostic sites dialog
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Silent/ diagnostic sites dialog
***Select a forward ORF and a set of
restriction enzymes before selecting the
menu item
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Silent/ diagnostic sites dialog
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Silent/ diagnostic sites dialog
Select diagnostic site if you are adding a site that is NOT in an ORF
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dCAPS dialog
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dCAPS dialog
Two orientations
WT or mutant cut
Red is the variant base
Green is a mutant primer base
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dCAPS dialog
Sequence of the DNA
Enzyme recognition sequence
Primer sequence
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Primers- hand designed
Drag the selection where you want the
primer
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Primers- hand designed
Drag the selection until the Tm is what
you want
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Primers- hand designed
Try to keep the %GC near 50%, if possible
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Primers- hand designed
Try to keep the %GC near 50%, if possible
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Primers- ApE designed
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Primers- ApE designed
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Primers- ApE designed
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Primers- ApE designed
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PCR reaction tool
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PCR reaction tool
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PCR reaction tool
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PCR reaction tool
Active primers show up here
Select this to activate just unique primers
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PCR reaction tool
Select primers to show them in the sequence
map
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PCR reaction tool
Click here to add selected primers as
new primer_bind features
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PCR reaction tool
Modify the priming search parameters
here
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PCR reaction tool
Select all of the class primers and sequences then copy
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PCR reaction toolPrimers can be:
sequencename (tab) sequencename (tab) sequence (tab) notefeature library format
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PCR reaction tool
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PCR reaction tool
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PCR reaction tool
Select oMBC22 and 23 primers.
These are the around the horn mutagenic
primers.
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PCR reaction tool
Select this to do a new PCR reaction
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PCR reaction toolIn primer pair mode, only one forward and
one reverse primer can be selected. Selected primers are in bold.
New PCR will become active when a pair of primers is selected.
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PCR reaction tool
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Gibson Designer
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Gibson Designer
Do oMBC26-oMBC27 PCR on pMLS280
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Gibson Designer
Do oMBC28-oMBC29 PCR on amilCP
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Gibson Designer
Do oMBC30-oMBC31 PCR on amilCP
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Gibson Designer
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Gibson Designeradd the three new DNA sequences in
orderIn this case, we don’t need new primers. We
are doing a Gibson using someone else’s
design
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Gibson DesignerFirst fragment
Second fragment
Press next to see each of the three junctions
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Gibson DesignerSecond fragment
Final fragment
Generate product
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Gibson DesignerThe first template sequence
The reverse primer sequence
The second template sequence
The forward primer sequence
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Gibson Designer
Design a new Gibson reaction
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Gibson Designer
Use the open plasmids with these selection
ranges
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Gibson DesignerFirst fragment
Reverse primer
Foreward primer
Second fragment
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Gibson Designerpriming hybridization
Gibson hybridization
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Gibson Designer
Adjust the TmAdjust the Tm
If there is already overlap between the templates, you
can use that
Adjust the overlap Tm
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Gibson Designer
You can also add non-templated sequence here
Set which fragment gets the overlap Add overlap to the other side
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Gibson Designer
You have to examine each junction
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Gibson Designer
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Gibson Designer
Gibson PCR products, primers etc. are in the file
comment
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Gibson Designer
Gibson PCR primers are in the file features
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Translation tool
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Translation tool
Translate all of a sequence, selected text, or any “CDS”
type feature
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Translation tool
Format the output
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Translation tool
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Enzyme Selection
Shift-click to change all tools to apply to
selection rather than sequence
You can select this option to change all
tools to apply to selection rather than
sequence
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Enzyme Selection
You can select this option to change all
tools to apply to selection rather than
sequence
This toolbar icon changes when you
have “selection only” applied
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Enzyme Selection
Current window to search
Apply search to selection
Current selection in window (can be
changed while the dialog is open)
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Enzyme Selection
All enzymes are shown here
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Enzyme Selection
The current enzymes can be changed by loading new ones
using the file menu WHILE THE DIALOG IS
OPEN
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Enzyme Selection
Click on any enzyme to select it
The enzyme currently under the mouse is
shown here
Shift-click on any enzyme to select only
that enzyme
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Enzyme Selection
The selection criteria are shown here
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Enzyme Selection
This is the count
This is the group
Any enzyme matching both the count AND
the group are underlined
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Enzyme Selection
Click here to select all underlined enzymes Click here to DE-select
all underlined enzymesClick here to de-select
ALL enzymes
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Enzyme Selection
Click here to select only enzymes that are currently selected AND
underlined
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Enzyme Selection
Right-click to search for enzyme info
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Enzyme Selection
Click here to do analysis functions
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Enzyme Selection
The difference group is special
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Enzyme Selection• Select the MCS in pMLS280• Open the enzyme selector• Select all unique enzymes in pMLS280• Chose the difference group and
pMLS280+selection within the difference selector
• Deselect the different enzymes• Do a graphic map• What do you see?
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Enzyme Selection
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Enzyme Selection
Find enzymes to clone amilCP into pMLS280
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Enzyme Selection• Select from 25 to 751 in amilCP
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Enzyme Selection• select all enzymes that are absent from
the selection in amilCP
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Enzyme Selection• select all enzymes that are ALSO present
somewhere in the plasmid, and in the Jorg collection
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Enzyme Selection• do a graphic map
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Enzyme Selection• do a graphic map
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Enzyme Selection• select all enzymes that are ALSO unique in the pMLS
MCS
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Enzyme Selection• select all enzymes that are ALSO unique in pMLS
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Enzyme Selection
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Preferences dialog
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Preferences dialog
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Recombination tool
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Recombination tool
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Recombination tool
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Recombination tool
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Graphic Map
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Graphic Map
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Graphic Map
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Graphic Map
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Graphic Map
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Graphic Map
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Graphic Map
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ABI files
ScaleDrag this to a sequence window to add the abi file
to a sequence file
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ABI files
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Align
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