10.1.09 Lab 3 - Bacteria Transformation

10.1.09 Lab 3 - Bacteria Transformation - So youll get...

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10/1/09 Creating the GFP fusion requires that we clone the cDNA into an intermediate cloning vector (step 1). Then we will move the cDNA into the binary vector for expression in plants (step 2). ** Our result from gel: backwards Understand this and write in notebook : recombinase was done by putting 2 plasmids and an enzyme together. Gateway Recombinase Cloning into the Binary Vector ccd gene – suicide vector – it makes a gene that kills the bacteria If any bacterium picks up a plasmid with a ccd gene, it will die. Selection of Correct Gateway clones: 1. ccdB gene encodes a poison 2. Kan gene makes cell resistant to kanamycin *but a screwed up plasmid can still have the Spec? gene w/o the ccdB gene. *the plasmid we started with already has GFP in it. *we get to select instead of screen. Start with a plasmid w/ spec and 2 strips of DNA And a plasmid with GFP, kan and ccd, 2 strips of DNA, Restriction enzymes will cut those strips and swaps them, but in opposite direction.
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Unformatted text preview: So youll get plasmid 1: spec and 2 swapped strips and ccd; and plasmid 2: w/ kan, GFP, 2 swapped strips. Materials 1. On ice: a tube containing your plasmid, the binary vector, and the recombinase reaction enzymes. 2. On ice: Bacterial (E. coli) cells that have been made chemically competent for plasmid transformations using calcium ion. 3. An agar plate with the correct anti-biotic mixed into the media for plating your cells. (pre-warming at 37 degrees). Incredibly complicated bacteria transformation protocol 1. Pipette 5 L recombinase reaction into 50 Ls of competent cells. 2. Place cells back on ice for ~5 min. 3. Pipette 50 L of cells+reaction onto a labeled agar plate. 4. Pour 15-25 sterilized glass beads gently onto plate. 5. Shake mercifully for at least 60 seconds. Take turns. 6. Dump beads out into plastic beaker. 7. Place plate back into at 37 degrees in the incubator....
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This note was uploaded on 02/17/2012 for the course BIO 1421 taught by Professor Farr during the Fall '08 term at Texas State.

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