Let Your Fingers Do the Walking -
Plan Your Next Subcloning
Written by: Erez Podoly, Nurit
You just received from your collaborator your gene of
interset (GOI = Gene
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:
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!
Get the complete sequence of pGEM from the site of Promega
(the company selling this plasmid).
Insert the sequence of pGOI into
pGEM according to the way pGEM-GOI was initially
Get acquainted with important elements within the plasmid
essential for successful subcloning.
Analyse the restriction enzymes cutting pGEM-GOI,
software to obtain further information about selected restriction enzymes.
Get the sequence of pQE-60 from the site of Qiagen
(the company selling this plasmid) and recognize important elements within
Use your common senseto decide
which strategy to adopt.
software to find several strategies to transform GOI
from pGEM-GOI to pQE-60.