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Welcome to 2. Gene Targeting

Cloning our gene of interest to add a green tag

Cloning our gene of interest to add a green tag

Aim: For our experiments we want to clone the collagen gene into a DNA plasmid that contains genes for green and red fluorescent proteins, which we will then insert into some mammalian cells. Last week I cloned the collagen gene into the DNA plasmid but I have mixed up my tubes. I now need to identify which tube contains the correct plasmid.

Method: I will use three restriction enzymes to cut each of the possible plasmids. I will then check the DNA fragments to identify the correct plasmid.

A plasmid is a short circular piece of DNA, ours is now ~7,000 bases long

Reminder of what the plasmid contains:
Fluorescent marker genes

  • Red Fluorescent Protein (RFP) = red coloured
  • Green Fluorescent Protein (GFP) = green coloured
    Antibiotic resistance gene
    Collagen gene (COL1A)

I can use the same restriction enzymes I used to insert the collagen gene. I can see the different sizes of the DNA fragments by running them on an agarose gel. The smaller fragments of DNA will move through the gel quicker and so will appear as bands lower on the gel whilst the bigger pieces will appear higher up.

Restriction enzymes are
like scissors for cutting DNA

Scientists use “bp” as a unit of measurement meaning the DNA is X base pairs long

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