RECOMBINANT DNA METHODS DAYS THREE TO FIVE

DAY THREE

Today, you will use agarose gel electrophoresis to analyze your PCR amplification from Day 3. This involves pouring and running a gel as you did on RDM Day 1. Today, however, you will run a standard 1% agarose gel instead of a low-melt gel. The protocols for pouring and running these gels are similar. You will also calculate the transformation efficiency of your AG1111 cells, use a "miniprep" protocol to isolate plasmid DNA from potential positives (inoculated on RDM Day 2.5), and set up restriction digests on this plasmid DNA.

DAY FOUR

You will use agarose gel electrophoresis for the third time today, this time to analyze your restriction digests from Day 3. Once you have identified which plasmid(s) are correct, you will transform the correct DNA into another E. coli strain, BL21. These cells have the machinery necessary to transcribe and translate the gfp DNA to create the Green Fluorescent Protein (GFP). The protocol for transformation is very similar to the transformation that you performed on RDM Day 2.

DAY FIVE

Today, you will see if you have successfully expressed GFP in E. coli BL21 cells, and will calculate the transformation efficiency of your BL21 cells.

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