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Unformatted text preview: MIT OpenCourseWare http://ocw.mit.edu 5.36 Biochemistry Laboratory Spring 2009 For information about citing these materials or our Terms of Use, visit: http://ocw.mit.edu/terms . Site-directed Mutagenesis and Transformation I. DNA Site Directed Mutagenesis A. PCR primer design II. Transformation (step 1 of cloning) A. PCR DNA RNA protein ( ) Central Dogma of Biology DNA segments carry the blueprints for protein synthesis. Figure by MIT OpenCourseWare DNA backbone and base pairs O O O O O O HO P P P P P O O O A T G C T O O O O O O OH P P P P P O O O H O CH 3 N H N HC C C C N C N H N CH N CH C N O Adenine (a) sugar-phosphate backbone C DNA backbone and base pairs O O O O O O HO P P P P P O O O A T G C T O O O O O O OH P P P P P O O O G A T A C H N H O CH 3 N HC C C C N C N H N CH N CH C N O Adenine (a) Thymine (t) 2 H-bonds H O H N N HC C C C CH N C N H N CH N C C N N H O H Guanine (g) Cytosine (c) 3 H-bonds sugar-phosphate backbone C DNA strands form an anti-parallel conformation O O O O O O HO P P P P P O O O A T G C T O O O O O O OH P P P P P O O O sugar-phosphate backbone G A T A C 5 The 5 end of a DNA strand terminates with a _________ group. phosphate The 3 end of a DNA strand terminates with a hydroxyl group. By convention, we write a DNA sequence 5 to 3 . A DNA single strand is deFned as a sense strand if the mRNA version of the identical sequence can be 5 translated to a protein. The compliment DNA sequence (the opposite strand) is called the antisense strand. DNA Cloning : in-vivo amplifcation oF DNA Recombinant vector with desired insert Abl K domain Antibiotic resistant vector 1. L igation 2. TransFormation Desired plasmid Chemi-competent E. Coli E. Coli transFormed with plasmid 3. Selection with Antibiotics without plasmid die Selection (ie. on an LB/agar plate with antibiotics) with plasmid survive and proliFerate with plasmid survive and proliferate In Modules 4 and 5, we are using E. coli cells for storage and expression. DH5 cells for storage. BL21(DE3) cells for protein expression. with plasmid survive and proliferate In Modules 4 and 5, we are using E. coli cells for storage and expression. DH5 cells for storage. BL21(DE3) cells for protein expression. For lab Session 2, you were provided with BL21(DE3) cells transformed with an H396P Abl(229-511)- encoding vector for protein expression. DH5 cells transformed with a wt Abl(229-511)-encoding vector for isolation of the wt vector DNA (by doing a miniprep). Cloning and Site-directed mutagenesis I. Overview of Molecular Cloning II. Ligation (step 1 of cloning) A. PCR B. Restriction Enzymes and Gene Insertion C. Session 3: Digestion to check for the Abl insert III. DNA Site Directed Mutagenesis A. PCR primer design B. Overview of the Quikchange strategy DNA Cloning: in-vivo amplifcation oF DNA 1. Ligation Recombinant vector with desired insert Abl K domain Antibiotic resistant vector 2. TransFormation Desired plasmid Chemi-competent...
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Slide2 11.45.23 PM - MIT OpenCourseWare http://ocw.mit.edu...

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