Unknown Plasmid

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Hoa Nguyen
Section 802
Final Lab report
Due December 12th, 2011
Transformation of Unknown Plasmid
Abstract: From the protocols that had been performed all semester, the purpose of this lab is to determine the identity of an unknown gene cloned in a plasmid. We created our own unknown plasmid using the Transformation method. We used methods such as a mini prep, quantification and sequencing protocols to observe and learn what this unknown plasmid contained. The results were not determined. Introduction: There are various reasons to transform DNA other than to help cure disease. Transformation of bacteria is also used to find out what that bacterium actually contains. A mini prep, quantification and sequencing protocols were performed. During the mini prep, we used bacterial cultures that contained the plasmid DNA which was growth the week prior. The purpose of this mini prep is to break open the cells and isolate DNA. The classic boiling method is used. Quantification is a fairly simple protocol. We quantify the DNA samples to get a more accurate reading of DNA. This involves the utilization of dyes that bind to DNA and emit a specific fluorescence upon binding. As the dye binding decreases, the amount of DNA increases giving the fluorescence to be approximately proportional to DNA concentration. Sequencing DNA involves cleaning up and separating and reading the sequence fragments from the previous lab. During the mini prep lab, a method used for sequencing was called the chain terminator method. (Wernick, 2011) Methods and Materials:

Transformation: Obtain two micro test tubes and label with one –plasmid and the other +plasmid. Place them in a foam rack and using a sterile pipette, transfer 250 microliteres of transformation solution which is the CaCl2 into each tube and place them on ice. Then scoop up a large colony of bacteria with a new sterile loop. With both tubes, add the colony into each and place them back onto the ice. With a sterile pipette,...
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