Editing the appearance of your sequence maps

By Chris | Published: April 18, 2016

Although we think that the default appearance of sequence maps in MacVector is very pretty, sometimes the defaults are not to everybody’s taste! If you think this way, then changing how maps look is very easy.

As usual with MacVector, there are many ways to quickly edit the appearance of a feature or multiple features. These all involve using the Symbol Editor.

  • Select the feature in the Map tab and go to OPTIONS > EDIT SYMBOLS FOR SequenceX.
  • Double click on a feature, in the Map tab
  • Select multiple features in the Map tab with the cursor, then double click somewhere in that selection.
  • Double clicking on the feature in the Tree View in the Graphics Palette.
  • Select one or more features in the Tree View and click the Edit button.

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    All of these options will open the Symbol Editor with the selected features highlighted. If you had selected multiple features, then some fields will be be shown in grey with a “mixed” symbol to show that the selected features had multiple values previously. If you enter a new value then all the selected features will be changed. However, if you leave that unchanged, then the symbols will be unchanged. This allows you to change just the colour, or some other attribute, of multiple features without changing all their other attributes.

    Remember though, that there are better ways of editing the appearance of multiple sequences, or changing the default appearance of all your files.

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    The Importance of Topology

    By Chris | Published: April 18, 2016

    MacVector understands the difference between circular DNA molecules and linear DNA molecules. Not just from the standpoint of how the molecule is displayed on the screen, but also in regard to how the analysis algorithms handle the junction at the ends of the circle. The Topology button controls the underlying linear versus circular state of the molecule. It is always shown on the toolbar of DNA/RNA sequence windows and can have two states.

    A great example of how this works is pBR322. This classic vector has its circular origin defined as the center of the EcoRI site that is traditionally placed at the 12 o’clock position on the map. You can use MacVector’s Graphics Palette to show the molecule in linear form. However, note that the EcoRI site is still displayed because although we are viewing the molecule as if it is linear, the Topology button is still set to circular, so the EcoRI site is still present.

    But if we click the Topology button, this changes the sequence from circular to linear so the sequence around the original EcoRI site goes from …xxxGAATTCyyy… to 5′-TTCyyy……xxxGAA-3′. The two halves of the site are now at opposite ends of a linear molecule, so no EcoRI site is present.

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    MacVector enforces this topology status when considering if a molecule can be displayed in circular form. It ONLY allows circular molecules to be displayed as a circle in the Map tab so that you don’t think you are working with a circular molecule when “under the hood” you are not. The solution is simple though – just click the Topology button!

    Read more.

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    How to increase the number of graphics levels to stop features overlapping.

    By Chris | Published: April 11, 2016

    MacVector tries to optimize the Map graphics layout using a trade-off between performance and minimizing unnecessary white space. Sometimes the default settings we have chosen are not ideal, particularly if you are looking at the Map tab of an Align To Reference window where you have a large number of reads overlapping the same region. To clean up these views, you can increase the number of graphical levels using the MacVector Preferences | Map tab.

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    For more information on the use of levels in the Map tab, please checkout this blog post.

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    Displaying CDS features as translations in the Map tab.

    By Chris | Published: April 11, 2016

    Where appropriate, features can be shown as residues when there is sufficient space to show them (for example when zoomed to residue). By default this is enabled for certain features, e.g. CDS features, genes, but it is controlled from the Symbol Editor and can be turned on/off for most features.

  • In the Map tab, double click on a feature to edit it.
  • Change the dropdown menu to Show as Graphic to disable this.
  • Select Show Residue Letters if Room to enable it.

    • For example, if this is enabled for a CDS feature when zoomed to residue the amino acid will be shown.

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    See this and this blog post for more details.

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    Calculating the melting temperature of PCR primers

    By Chris | Published: April 11, 2016

    Calculating an accurate melting temperature of your oligos, your template and of the predicted product is important to set the cycling parameters of your PCR machine.

    To test a pair of primers, use Primer Design (Primer3).

  • Open your template sequence
  • Run ANALYZE > PRIMER DESIGN/TEST (Pairs)
  • Click on the Use This Primer checkbox for both the left and the right primer
  • Add your primers

    • To test a single primer use Quicktest Primer

  • Open your template sequence
  • Run ANALYZE > QUICKTEST PRIMER (Individual)
  • Paste in your primer sequence

    • QuickTest Primer shows Santa Lucia, Breslauer and Baldino estimates of the Tm.

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    We always strive to use the most up to date algorithms to calculate Tm:

    MacVector uses the combined thermodynamic parameters of Santa Lucia (PNAS 95:1460-1465, 1998) which have become widely accepted as the most accurate values.
    MacVector makes adjustments for Mg++ and dNTP concentrations in the reaction mixture as described by (Ahsen et al, Clinical Chemistry 47:1956-1961, 2001).

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    Removing a restriction site from a plasmid with the Cloning Clipboard

    By Chris | Published: April 11, 2016

    Being able to remove a restriction site by digesting with an enzyme that cuts at one site to linearize a plasmid, Klenow treating the linear fragment, then religating the fragment, is a useful trick in the lab. It can help you produce a suitable, and simple, cloning site when none previously existed in your vector. It is very simple to recreate this in your vector sequence using the Cloning Clipboard.

  • Select the restriction enzyme recognition site you want to remove.
  • Click DIGEST to linearize the plasmid and place it on the Cloning Clipboard.
  • Switch to the Cloning Clipboard and select your new fragment.
  • Click the Circularize button.
  • Modify the ends to blunt them both using the Fill or Cut Back buttons.
  • Click Ligate.

    • A new circular sequence will be created from your digested fragment.NewImage

    The origin of your plasmid will have changed to the digested site, so you may want to rotate the sequence to match the original sequence.

    Read more.

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    The simplest way to export high quality graphics from the Map tab

    By Chris | Published: April 4, 2016

    So you’ve finally go the graphics for that new construct looking absolutely fabulous in MacVector and you want to get it into an image editing program (e.g. Adobe Illustrator, Photoshop or even PowerPoint) ready for that critical presentation. How best to do it? The easiest way, by far, is to simply choose Edit->Copy to copy the image to the clipboard, then switch to the target application and choose Edit->Paste. Some applications, such as the popular Preview.app that is installed on all Macs in the /Applications/ folder, even have a handy File->New From Clipboard menu item:

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    There is no need to “select” the graphics you want to copy, the entire image will always be copied in high resolution “vector graphics” so that you can blow up the resulting image to any size with no loss of resolution. If you create a New From Clipboard window in Preview.app, you can then “Save As…” the image in a variety of different formats.

    There are other approaches to getting graphics out of MacVector – with recent versions (13.5 and above) the File->Export Tab Contents As… menu item lets you export graphics in a variety of different formats. If you need the graphics broken out into multiple pages, then use File->Print and choose the PDF->Save To File… option.

    You can learn more about this from this 101Tips post.

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    Keep up to date with MacVector auxiliary files

    By Chris | Published: April 4, 2016

    MacVector has a large collection of data files that are installed in folders alongside the MacVector application. These include Restriction Enzymes files, Scoring Matrices, documentation, tutorials, a variety of common cloning vectors and even a few useful Applescripts.

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    If you use MacVector’s built-in automatic updater to install each new release and bug-fix, you will find that only the application itself gets updated. However, with each new release we are constantly updating and revising these auxiliary files. For example, for MacVector 14.0 we updated the Restriction Enzyme files and also added DAM and DCM methylase affected sites so that you can easily spot sites that would be blocked by either of these methylases. We added additional vectors and Applescripts, and updated tutorials and documentation. You can download a zipped archive of the current set of auxiliary files from our website.

    Download and extract the files, then copy and paste the individual folders into your current active MacVector folder. You can always find the current set of auxiliary files on our downloads page.

    You can also use the full installer to replace your entire active MacVector folder with the latest release – that can always be found here.

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    Using BLAST to automatically annotate a sequence

    By Chris | Published: April 4, 2016

    You can use the Database->Auto-Annotate Sequence function to quickly annotate a bare sequence using existing annotated sequences on your file system. However, this only works if your collection of sequences contains features representing all parts of the bare sequence. Luckily, if you have an unannotated region after running Auto-Annotate, you can use MacVector’s built-in BLAST function to find and download matching annotated sequences, then run Auto-Annotate to add features to the blank region.

    If you are interested in this, the full steps are discussed in this blog article.

  • Briefly, the first step is to select the unannotated region of your test sequence. You can do this in the Map tab by choosing the sequence selection mode in the floating graphics palette and drag-selecting the blank region.

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  • Choose Database->Internet BLAST Search to find the best hits to the blank region.
  • Select the top four or five hits in the BLAST Results Description List and choose Database->Retrieve To Folder.
  • Now run Database->Auto-Annotate Sequence, choosing the folder where you retrieved the matching hits.
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    How to select all identical restriction enzyme sites

    By Chris | Published: April 4, 2016

    The Automatic Restriction Enzyme Analysis tool that is displayed on the sequence in the Map tab is very powerful. It automatically displays a custom set of restriction enzyme recognition sites on every sequence that you open. Unique recognition sites are displayed in red and sites with two or more cut locations are displayed in blue. Those sites also show the total number of cut locations of that recognition site.

    To select every site of a single enzyme just double click on a single site and every other site will also be selected. Additionally the Graphics Palette will now show those enzyme sites in the Results tree view.

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    To change the Automatic RE Analysis options open MACVECTOR > PREFERENCES and switch to the MAP tab. Read more about restriction mapping.

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