Visualising ORF analysis results in the MAP tab.

By Chris | Published: November 25, 2015

The Map tab of MacVector is a powerful way to visualise and interact with your sequences. All analysis tools will work directly in the Map tab. You can design primers, ligate and digest fragments from the Cloning Clipboard, visualise translated CDS regions and much more. In fact one of the only tasks, you will need to switch to the Editor tab for, is to manually type in some sequence. …and who needs to do that nowadays!

As well as the Map tab, you also have the Map tab in the Results window that is produced by most analysis tools. For example when designing primers you get a graphical map of your primers and products.

The Map tab of a Results window is as flexible as the Map tab of the main sequence window. If you need to Zoom to Residue to see your sequence results at the sequence level, or Drag to Zoom to show just a small areas, then go ahead. The Graphics Palette acts exactly as it does with the main sequence window’s Map tab.

A nice way to visualise an ORF analysis results is via the Map tab, except zoomed to the sequence level, rather than using it as a graphical overview of the ORF analysis.

When you initially run ANALYSE > ORF ANALYSIS the Map tab in the results window shows the entire sequence, with graphical symbols representing ORFs.

SequenceSample nucl Results

However, if you click ZOOM TO RESIDUE in the Graphics Palette, then instead you will see your sequence, any annotated features, and the amino acid sequence of any ORFs in your sequence. It’s a powerful way to visualise the results.

Graphics Palette and SequenceSample nucl Results

You can also directly analyse the results within the Map tab. Just click on an ORF and copy that sequence to a new one, or do a Blast search directly on that sequence to see if it’s the coding region you are looking for.

  • Run ANALYSE > ORF ANALYSIS
  • Choose the appropriate settings and click OK
  • Switch to the Map tab in the Results window
  • Click Zoom to Residue in the Graphics Palette
  • Select the appropriate ORF
  • You can either copy it and use FILE > NEW FROM CLIPBOARD to create a new sequence, or DATABASE > BLAST to search for it.

    • Remember that if you want to view both the Map tab and the Text tab you can drag the tab from one window and view both together.

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    Cloning Clipboard: Documenting the history of a construct

    By Chris | Published: November 16, 2015

    Designing and documenting cloning strategies is easy with the Cloning Clipboard. You can perform quite complex ligations by simply dragging compatible ends together. Not only that but every step is documented and recorded in the resulting sequence, so you always know where each fragment came from.

    NewImage

    Ligating a single fragment into a vector is as straightforward as selecting two restriction sites and using EDIT | DIGEST, then selecting target sites in a vector and then EDIT | LIGATE. Every digestion places the fragment on the Cloning Clipboard and you can ligate these fragments by clicking on one end and dragging it onto the end of a different fragment. It fully understands compatible overhanging sticky ends preventing you from accidentally creating biologically impossible molecules. It even allows you to fill or cut back ends to make them compatible.

    However, when you open this construct a year later, you don’t want to have to look back in your lab book to know how you generated it. Neither do you want to have to email a lengthy description every time you send a plasmid to a colleague. MacVector does all this for you and fully documents the cloning strategy that you used. Every single ligation is documented as a FRAG feature which is annotated to the target sequence. This is regardless of whether you used COPY | PASTE for a single fragment, or whether you ligated multiple fragments on the Cloning Clipboard and ligated that fragment into a cloning vector. All FRAG features from previous ligations are preserved in new ligations. So if you joined three fragments together on the Cloning Clipboard, then ligated those fused fragments into a vector you would have four FRAG features documenting exactly what you did.

    As well as the source of the fragment and timestamp, the enzymes used to digest the fragment, and any modification of the overhang are also recorded. So if in the aforementioned scenario if you had Klenow treated a fragment to remove an overhang, that would be documented in the FRAG feature for that fragment.

    PBR322 nucl Features

    By default FRAG features are hidden, but you can easily make them visible to visualise the history of a construct by selecting the FRAG feature in the Graphics Palette and making them visible.

    Graphics Palette and Analyses and pBR322 nucl Map

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    101 things you (maybe) didn’t know about MacVector: #47 – Optimal PCR Annealing Temperature

    By Kevin | Published: November 11, 2015

    I wrote about how MacVector calculates the melting temperature of a primer in an earlier blog post. One other common question we get is, “once I have designed a pair of primers, what is the optimum annealing temperature to use in the PCR?” MacVector calculates this and displays the results in the Analyze | Primer Design (Primer3) spreadsheet results tab;

    Primer3TableResults

    Here, the Tm of both the left and right primers is calculated using the Santa Lucia thermodynamic nearest neighbor algorithm. The Tm of the product is also displayed – because the product is nearly always longer than 35 bp, we use the Baldino algorithm. Finally, the recommended annealing temperature (Ta) for use in the reaction is listed in the right-most column.

    This calculation takes into account the Tm of both the primers AND the product, based on the algorithms described in W. Rychlik, W.J. Spencer, and R. E. Rhoads, Nucl.Acids.Res. 18:6409-6412(1990). They determined empirically that the greatest yield was observed in a PCR when the Ta was calculated according to this simple formula;

    0.3 x Tm(primer) + 0.7 x Tm(product) – 14.9

    where the lowest Tm of the two primers is used in the calculation.

    Many scientists use a simple rule-of-thumb where they calculate a Ta by subtracting 10 from average Tm of the two primers – you can see this will often give a similar result to MacVector’s calculation. However, the importance of the product Tm in the Rychlik paper explains why sometimes MacVector will recommended a Ta that might be much higher or lower than you would expect based on the primer Tm. In particular, if the chosen primers are of much lower G+C% content than the product, it can be important to maintain a higher annealing temperature than might be expected to prevent the product from annealing with itself and blocking the primers from binding.



    This is an article in a long running series of tips to help you get the most out of MacVector. If you want to get notified every time a new tip gets published, follow us @MacVector on twitter (or check the feed for the hashtag #101MacVectorTips) or like us on Facebook.

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    MacVector 14.0.4 is OS X 10.11 El Capitan compatible.

    By Chris | Published: October 7, 2015

    OS X 10.11 El Capitan was released last week. We’ve been testing MacVector on the developer releases of OS X EL Capitan in the run up to the official release. However, as usual we prefer to fully test on an official release of OS X before saying that MacVector is supported. We’ve now done so and we are pleased to say that MacVector 14.0.4 works fine on El Capitan. Neither have we had any reports from the MacVector user community, so we are happy to say “upgrade away”!

    If you are running older versions of MacVector then why not upgrade? As well as being EL Capitan compatible you’ll get all these new features. Request an upgrade quote if that tempts you.

    In other update news there will be a MacVector 14.0.5 minor update release within the next couple of weeks with a few bug fixes and MacVector 14.5 will also be out soon (wait until you see the new Agarose Gel tool!).

    ElCapitan

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    Buy two copies of MacVector 14 for the price of one.

    By Chris | Published: September 30, 2015

    For the month of October if you buy a personal or standard license of MacVector Pro then you’ll get one free!

    We never stop working to keep MacVector your best solution for designing primers, subcloning into vectors, aligning your sequences and checking small sequencing projects on the Mac. So don’t miss this chance to start using the best sequence analysis application for the Mac, and with two copies for the price of one, you can let your colleagues start using it too.

    If your lab has two Macs, then purchase two personal licenses at 50% of the cost! That’s a license per Mac at a very good price. If you have more than two Macs, then you can purchase two standard licenses for the price of one instead. You’ll be able to install the two standard licenses on all the Macs in your lab and also at home or on your laptop when you are away at a conference.

    Request a quote now using the promotional code AGAROSE-GEL

    Don’t forget that these are perpetual licenses and include a year of updates too!

    If you take advantage of this offer then you’ll receive MacVector 14. This is the current release and is a 64 bit application that’s fully supported on all 64 bit Macs running OS X Snow Leopard to OS X El Capitan. It’s also got useful new tools as well such as the Primer database. However, because you get a year of updates included it means that you will get MacVector 14.5 when it is released later this year. Normally we wouldn’t mention a upcoming release before it’s out, but we are very excited about MacVector 14.5 because of the new Agarose Gel tool. You will be able to view simulated gels right in MacVector that are so realistic that you’ll wonder “Is it real or is it MacVector?”.

    Gel and Cloning Clipboard

    We’ve worked hard to bring you MacVector 14 and we really hope that you’ll take advantage of this offer. If you are not convinced then download the trial now. However, do not forget that this attractive offer will only be valid until the end of October. These prices are for purchasing academic standard and personal licenses of MacVector Pro. Please ask for a quote for cloning, network, commercial or Assembler licenses. You’ll get an equivalent discount.

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    Using the Primer Database to store your lab’s collection of primers

    By Chris | Published: August 21, 2015

    MacVector now directly supports storing primers within a primer database. The Primer Database tool allows you to save and retrieve primers from subsequence files within Primer3 and Quicktest Primer. You can easily create your own primer database, use existing files or import primers from Excel.

    Many users use subsequence files to store primers, and in fact we have a tool that allows you to convert an Excel spreadsheet into a subsequence file full of your primers. Some of these users have asked whether we could make this easier and also better integrate it better into primer design within MacVector.

    So in MacVector 14 we have introduced the Primer Database tool. The new tool is integrated within all the primer design tools and allows you to save primers directly from the multiple primer design interfaces to the primer database. You can also scan your sequences with the Primer Database.

    The Primer Database is backwards compatible with subsequence files so users, already using subsequences to store their primers, can seamlessly migrate to using the Primer Database.

    Primer Database

    For users, new to storing primers, there is a new Primer Database.nsub file (installed in the /MacVector/Subsequences/ folder) populated with a number of common universal primers. This is used as the default database file, but you can easily choose any file of your own, or add your own primers to this file, or to a copy of it.

    Primer Database Search – this is a new function in the Analyze menu. It let’s you scan sequences for potential primer binding sites against the Primer Database. It is similar to the Nucleic Acid Subsequence search function, except that it uses Primer Database.nsub as the default search file and has extra settings to simplify handling primers with tails and/or mismatches to the target sequence.

    To use the Primer Database Search:

    • Open your sequence
    • Select ANALYZE > PRIMER DATABASE SEARCH
    • Choose any parameters and click OK

    Remember that you can easily annotate any result back to your sequence by dragging and dropping from the Results window.

    Quicktest Primer – this now lets you save designed primer direct into the current primer database and also lets you retrieve primers from the database via a simple popup scrolling menu. In addition, this interface now handles primers up to 200 nucleotides in length.

    To save a primer from QT Primer:

    • Open ANALYZE > QUICKTEST PRIMER
    • Design your primer
    • Click ADD TO…
    • Give the primer a name and add a comment. Click OK

    QT primer Database

    Primer Design (Primer3) – you can now select primers in the spreadsheet result window and add them to the Primer Database and select primers from the database using the popup scrolling menu.

    To save a primer from Primer Design:

    • Open ANALYZE > Primer Design (Primer3)
    • Design your primer pair
    • In the spreadsheet right click on a primer
    • Choose ADD PRIMER TO DATABASE
    • Give the primer a name and add a comment. Click OK

    In both interfaces the popup menu is accessed via a new menu.

    Screenshot 21 08 2015 12 16

    Additional changes have been made to the graphical representation of primer binding sites in both the Primer3 and Primer Database Search results so that the actual sequence of the primer (complete with mismatches and/or tails) is displayed when zoomed to the residue level and if you select and copy the product graphic, the actual product sequence is placed on the clipboard which you can paste into a new document.

    All of these new features support mismatches and restriction site tails.

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    101 things you (maybe) didn’t know about MacVector: #46 – How to increase the number of graphics layers in the Map tab

    By Kevin | Published: August 4, 2015

    The graphical maps of heavily annotated sequences can get busy in a hurry. You may end up with so many features overlapping a specific location on a sequence that the graphical images for those features “pile up” on top of one another at the top or bottom of the display. While this can happen with heavily annotated sequences, it is most often encountered with Reads in an Align To Reference window, something like this;

    Overlapping Features

    The reason this occurs is because MacVector tries to ensure that the Map graphics are drawn in a reasonable amount of time and also wants to ensure that the maps do not have an inordinate amount of white space. Accordingly, it limits the number of “layers” of graphics to be displayed. If a graphical item will not fit in the available space without overlapping another item, it is placed on the uppermost (or lowermost) line, where you will often see features “piling up” on top of each other. To fix this, you can adjust the number of layers using the MacVector | Preferences | Map View panel and changing the Maximum Levels setting;

    Map View

    Changing this to 30 cleans up this particular display;

    OverlappingFeaturesFixed



    This is an article in a long running series of tips to help you get the most out of MacVector. If you want to get notified every time a new tip gets published, follow us @MacVector on twitter (or check the feed for the hashtag #101MacVectorTips) or like us on Facebook.

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    Editing the appearance of individual sequences maps

    By Chris | Published: July 21, 2015

    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.

    Rather than edit the appearance of all of your sequences it is far better to modify the default symbol set*. Then, when you create a new sequence, or import a Genbank file, its appearance will be exactly how you like it! Not only that but with the Auto Annotation tool there is an easy way of scanning your sequences against a template to ensure their appearance is the same.

    However, sometimes you really do need to change how a single Map looks. 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. To do this:

  • 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 sequences in the Tree View and click the EDIT button.

    • Editing Symbols

    Graphics Palette 2

    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 that you can restore the shipping defaults for a symbol at any time by clicking the Shipping Defaults button in the Symbol Editor. Also remember that to edit the default symbols for all new sequences ensure no sequence is open or hold down the OPTION key and go to OPTIONS > DEFAULT SYMBOLS… This will open the Default Symbols Editor which is difference in appearance to the Symbol Editor for sequences.

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    MacVector Cloning Edition

    By Chris | Published: July 14, 2015

    Does your lab only need a simple DNA analysis application that will help you design primers and cloning experiments? Has your budget been cut to the bone? If the answer is a resounding “yes”, then we’ve released a new version of MacVector just for you.

    MacVector Cloning Edition provides all of the functionality you need to design and document clone construction experiments in an affordable package that has the high quality and ease of use that our customers have come to rely on. While MacVector Pro Edition contains a wide variety of tools and functionality, many users don’t need all of the ‘bells and whistles’.

    Here’s what you can do with the MacVector Cloning Edition:

    Primer Design

    Get rid of a hairpin or primer dimer by sliding your primer along the template. Add restriction sites or even a mismatch to introduce a new silent site. Alternatively select a gene and get a ranked graphical list of the best primer pairs to amplify it. Drag and drop annotation of primers.

    Primer Database

    The Primer Database tool allows you to store primers direct from MacVector. You can scan sequences for potential primer binding sites against the Primer Database including mismatches and restriction site tails.

    Quicktest Primer

    Auto Annotation

    Annotate blank sequences with a few mouse clicks or curate your own lab’s sequences so that they all appear the same.

    Ecoli nucl Map

    Graphics

    Display beautiful feature rich maps with a sequence overview allowing you to see your full sequence at a glance. Export publication quality maps of your sequences.

    Cloning

    Cloning Clipboard

    Digest a fragment, then drag and drop on a vector to ligate it, or drag multiple fragments together. Clone with digested fragments, Gateway Cloning, TOPO cloning or Gibson Assembly). Constructs are fully documented.

    Restriction Mapping

    Display only your lab’s freezer drawer of enzymes dynamically on your sequence. Flexible searches show only enzymes that cut in one location and not in another. 

    Visit our website to see the full functionality available in MacVector Cloning Edition and the differences between the editions.

    MacVector Cloning Edition costs $475 for a perpetual academic license for one Mac. It includes a year of updates but the license itself will never expire. If you need the extra functionality in MacVector Pro, then you can upgrade at any time for the difference in cost. There is no penalty for trying the Cloning Edition first.

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    MacVector for Windows update

    By Chris | Published: July 14, 2015

    The development for MacVector for Windows progresses. It’s not ready for release yet but it’s definitely the last stage before testing.

    MV4WIN cloning Clipboard

    MacVector for Windows is not a Java version of MacVector. MacVector, the original, is designed for, and fully integrates with, the OS X environment. We know users really appreciate that. If you can use a Mac, you can analyze your sequences with MacVector! So we wanted MacVector for Windows to be the same native fully integrated application. Easy to use for anybody familiar with Windows.

    MV4WIN Ligation Dialogue

    Even though it’s a Windows application MacVector for Windows will still be familiar to anybody who’s used it on the Mac. All the usual features and tools are there.

    MV4WIN Desktop

    There will be a MacVector Free version of MacVector for Windows available for download during 2015.

    If you want to be kept updated about MacVector for Windows then either follow us on Twitter or Facebook or email Support and ask us.

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