Let Your Fingers Do the Walking -
Plan Your Next Subcloning

Written by: Erez Podoly, Nurit Kleinberger-Doron

You just received from your collaborator your gene of interset (GOI = Gene Of Interest) cloned in the plasmid pGEM. You wish to transfer GOI from pGEM-GOI to pQE-60, since pQE-60 contains elements required for induced expression. This is illustrated below:

Planning this subcloning is the subject of this tutorial.

As you'll see, there are several ways to perform the subcloning, and some are better than others.  Using software available on the web, you'll try to find the best strategy. In order to do that, you'll perform the following steps:

At  Home

  1. Get the complete sequence of pGEM from the site of Promega (the company selling this plasmid).
  2. Insert the sequence of pGOI into pGEM according to the way pGEM-GOI was initially constructed.
  3. Get acquainted with important elements within the plasmid essential for successful subcloning.
  4. Analyse the restriction enzymes cutting pGEM-GOI, using WebCutter software.
  5. Use REbase software to obtain further information about selected restriction enzymes.
  6. Get the sequence of pQE-60 from the site of Qiagen (the company selling this plasmid) and recognize important elements within this sequence.
When performing this assignment, you'll have to fill some data in the accompanying form. This data is essential for the class session. Bring the filled forms to the class!

In  Class

  1. Use CloneIt software to find several strategies to transform GOI from pGEM-GOI to pQE-60.
Use your common sense to decide which strategy to adopt.
Go to the home assignment
Go to the class assignment