2010 Bio 311 Lab B Manual (part 3)

2010 Bio 311 Lab B Manual (part 3) - B5. Bacterial...

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67 ©1999 Lodish, Molecular Cell Biology B5. Lab Overview: You will see two different methods of bacterial transformations today your bacteria. Your Lab Instructor will transform ElectroMAX DH5a-E bacteria with an experimental ligation mixture using the electroporation method. Then you will transform commercially available competent BL21 (DE3) bacteria by the traditional method. You will incubate the bacteria with your some of your ligation mixtures or with the control plasmid. Then you will plate the transformed cells on the kanamycin-containing (KAN) LB agar plates that you prepared during lab and on provided ampicillin-containing (AMP) LB agar plates. The competent bacteria will die on KAN plates unless they have taken up a plasmid with a KAN resistance gene during transformation. incubate the plates overnight at 37 0 C to allow the transformed bacteria to grow. Next time, you will analyze the resulting clones by “colony PCR”. Background E. coli bacteria do not take up DNA as efficiently as other bacterial strains. The ability of bacterial cells to take up DNA is called “competency”. The bacteria become “transformed” by the new DNA. Transformed bacteria can be turned into protein factories to express any gene placed in them on the appropriate vector. Plasmids are common vectors used to put genes into bacteria. Plasmids are small circular pieces of DNA that replicate autonomously in the cytoplasm of the bacterium. Bacterial cells can be induced to take up plasmid DNA by two main methods, use of divalent salts (such as CaCl 2 ) and application of voltage (electroporation). We are going to use commercially available competent cells for the transformations in this lab and follow the manufacturer’s protocol The classic methods for making competent bacteria and transforming them are is described in DNA Science, Micklos & Freyer (2003) on pages 457 to 468 and in Molecular Cloning, A ± Read "An Overview of the BCD labs" and Lab B5 . ± State lab Goal(s) ± Outline or make a flow chart of the methods. ± Complete all calculations. ± Answer Quiz Questions
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68 Laboratory Manual, Sambrook & Russell (2001) pages 1. 24-26. In general, salt competent cells are made by washing the cells with 0.1M CaCl 2 or other divalent salt mixture. The Ca ++ is hypothesized to neutralize the repulsive negative charges on the cell membrane as well as on the DNA. Treatment of the competent cells for a brief period at 42 0 C, called a heat shock , increases the permeability of the cell membrane and thus DNA uptake into the bacterial cell. Following heat shock, media is added to the cells, and the cells are incubated for a short period at 37 0 C. This allows the transformed cells time to express the genes on the plasmid, such as the antibiotic resistance gene. Subsequently, when the cells are plated on antibiotic-containing media, only the transformed cells grow. Electroporation is the application of high voltage to cells to force DNA into them. The method was introduced to transform eukaryotic cells (also called transfection) and delivers higher transformation efficiencies.
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This note was uploaded on 03/28/2011 for the course BIO 311 taught by Professor Staff during the Spring '08 term at SUNY Stony Brook.

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2010 Bio 311 Lab B Manual (part 3) - B5. Bacterial...

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