Recombinant DNA Methods Days Two and Two Point Five

RECOMBINANT DNA METHODS DAYS TWO AND TWO POINT FIVE

BACTERIAL TRANSFORMATION

On Day 1, you attempted to ligate (join) your pET vector fragment with your GFP insert. Today, you will introduce the ligation mixture into AG1111 E. coli cells that have been made competent for transformation. Here are a few pieces of equipment/reagents used in this protocol.


You will be performing your transformations in Falcon 2059 tubes. These tubes are made of polypropylene and hold 14 ml.	You will melt your ligations in a thermocycler set to 65C. The thermocyclers will also be used in the PCR amplification experiment you will perform at the end of the day today.

You will "heat shock" your competent cells in one of these silver water baths (set to 42C). They can be found at benches D1, G3, and L1.

To make it easy to find your tubes in the roller drum, label the top of each cap with a piece of tape with your bench letter on it.

POLYMERASE CHAIN REACTION (PCR)

Today, you will begin your PCR experiment to identify the location of your transposon insertion in your Ara- strains from the Microbial Genetics module. Specifically, you will isolate chromosomal DNA from wild type and Ara- strains, then perform PCR amplification using ara and lacZ (transposon) gene-specific primers.


You will lyse your E. coli cells to obtain chromosomal DNA by heating samples in a 90-95C heat block.	Your TAs will deliver special thin-wall PCR tubes to your bench. These blue 0.5 ml tubes are pictured here (left photo). (The right photo is of a 1.5 ml Eppendorf tube.)

SCREENING TRANSFORMANTS AND INOCULATION OF POTENTIAL POSITIVES (DAY 2.5)

On Day 2.5, you will be screening your classmates' plates to determine if their transformations were successful, and will be inoculating overnight cultures for them for use on Day 3.

Here is a photo of a "vector + insert" transformation plate generated by the 7.02 staff during our "Run-Through Week." Each "colony" consists of millions of cells, each containing the same piece of plasmid DNA. Some of these colonies are made up of cells with your pET-GFP plasmid!

You will need to use sterile technique when you inoculate your cultures today. Look over the pictures from GEN Day 1 to refresh your memory!

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You will "heat shock" your competent cells in one of these silver water baths (set to 42C). They can be found at benches D1, G3, and L1.	To make it easy to find your tubes in the roller drum, label the top of each cap with a piece of tape with your bench letter on it.