• Top five considerations for ORF selection

    What to think about when ordering an ORF construct

    What's New - Getting the right ORF does not have to be difficult!

    With so many options available to you, how do you know how to pick the right clone(s) for your over-expression project? Whether you are planning to perform an RNAi rescue experiment or want to screen an entire pathway, selection of the right reagents can mean the difference between a smooth path to your next publication or months of frustration.

    1. Check the accession number

      Searching by Gene ID and Gene Symbol are great ways to find the most reagents for your gene, but don’t forget to check the accession number to ensure the clone you pick matches the nucleotide sequence and splice form you require.

    2. Select the best format for your needs

      Do you need an expression-ready lentiviral format? Are you planning to put the construct into different vectors and utilize it in multiple cell lines? These are the questions you need to ask to determine what is the right format for your experiments. If you want to be able to put the construct into many different vectors, a Gateway® compatible clone might be most helpful. On the other hand, if you need to develop a stable cell line, a lentiviral vector, or even premade lentiviral particles might save you considerable effort.

    3. When multiple similar formats exist, choose the one that matches the most requirements

      You can always move the ORF from an entry vector into a specific destination vector or you can supplement your library with a similar format that contains unique constructs (e.g. supplement the ORFeome Collaboration Collection with selected Human ORFeome v8.1 clones.

    4. Sequencing

      Over-expression constructs are fully or partially sequenced by the source lab(s) that created the collection and are carefully monitored thereafter. However, even under the most ideal circumstances the constructs are subject to errors; it is always best practice to streak and sequence at least three colonies to ensure you are working with a pure population prior to beginning your experiments. A little work upfront can save a lot of frustration later.

    5. Know your scope

      How many clones do you require? Are you looking to examine just a few select genes, or are you trying to study a pathway, gene family, or even looking at a whole genome screen? Understanding your scope can help ensure you have the right resources in place, the right budget, and can plan your experiments properly to move forward as efficiently as possible. Knowing what you really need can ensure you are looking at the right collection options for both your short-term and long-term experimental requirements. For example; if you want to buy a larger library, but do not want to sub-clone, you probably want to look at a lentiviral format rather than an entry clone.

    • Bonus - Buying vs cloning

      One of the most important considerations in this decision is the cost of time. If a construct is available for purchase, it is a very simple process to order it and it will usually arrive within a few days to a week depending on where it has to be shipped. If one wants to clone a construct de novo, there are many steps including the design of primers, isolation of the gene region, cloning, and sequencing for confirmation. All of these steps take considerable time and resources away from what is, perhaps, a more experimentally valuable task.

    Additional Resources

     

Share: