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Week Eight

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  The next step to perform transformation is to collect the plasmid. E. coli containing pRAD1 was streaked on 50 ug/ml ampicillin LB plates and placed in the 37-degree Celsius incubator. After 24 hours in the incubator a gram stain should be performed to ensure the correct bacteria has grown; E coli, depicts gram negative rod s. A 20 ml 50 ug/ml ampicillin LB broth flask is inoculated with a colony from the plate and placed in the shaking incubator. The next day it is ready to be used for plasmid extraction. A procedure that breaks open the cell membrane, releasing genetic information , and discards the cell debris.   Materials:   -Microcentrifuge tubes   -Centrifuge   - pRAD1 E. coli culture   - Zymo research plasmid extraction kit     Protocol:   1. Add 600 ul of bacterial culture to a 1.5 ml microcentrifuge tube.   2. Add 100 ul of lysis bu ff er and mix by inverting the tube 4-6 times.   a. The next step must be c...
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Week Seven

  This week marks the start of the transformation process of D einococcus caeni . Before we are able to perform the transformation procedures there are a number of things that must be prepared. Transformation occurs naturally in the environment under selective pressures that cannot accurately repli cated, thus the bacterial cells ability to be transformed must be included . We call these included cultures comp etent cells . Firstly, competent cells of   the bacteria in focus must be prepared. The comp etent cell are chemically prepared in the lab. Sodium chloride, or salt, is used to neutralize the negative charge of DNA and the phospholipid bilayer in the cell membrane, promoting the DNA to move closer to the cell. The cells are stored in the –80 degrees Celsius freezer to maintain the permeability of the cell membrane while the glycerol prevents the cells from freezing and lysing.     Materials:   -TGY broth   -Glycerol 60%   -DI wate...
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