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Unformatted text preview: Biomed I Lab # 7 Bacterial Transformation Objectives To become familiar with the structure and use of plasmid DNA To transform competent DH5 cells with plasmid DNA. BIOHAZARD
THE NEXT FOUR LABS INVOLVE THE USE OF BACTERIA AND CARE SHOULD BE TAKEN IN THE HANDLING AND DISPOSAL OF ALL INSTRUMENTS USED IN THESE LABS-----WASH HANDS THROUGHLY BEFORE AND AFTER EACH LAB Introduction Genetic information is stored as strings of deoxyribonucleic acid (DNA) molecules Each gene sequence encodes for specific protein as and when required by the cell Most cells in the organism have the same genetic information but vary in protein content. Introduction Most of the genetic information is stored in chromosomes . Higher organisms have several chromosomes (46 in humans). Prokaryotic organisms (bacteria) have a single chromosome Bacteria also have non chromosomal DNA known as plasmids
DNA coding Gene 1 Non coding DNA DNA coding Gene 2 Higher organism DNA http://www.west.asu.edu/lsi/sum04/wk4/PLASMID!.jp Antibiotic resistance Plasmids carry genes that are essential for bacterial survival in hostile environment Example: Antibiotic resistance gene for antibiotic degradation. Antibiotic resistance genes encode enzymes that degrade the antibiotic and prevent it's action. Because the antibiotic is destroyed, the antibiotic resistance cells are able to grow and replicate in the presence of a antibiotic. Biotechnology The plasmid can be readily transferred into bacteria by the process known as transformation. However the bacterial cell must have a weakened cell wall for plasmid entry (made competent). Plasmid DNA can be easily manipulated for preparing large quantities of DNA or protein Recombinant Insulin is manufactured by inserting the human insulin gene DNA into a plasmid and transforming it into bacteria. Non competent bacterial cell Chromosomal DNA plasmid Competent cell Tranformed cells `T" Non- Tranformed cells `C" Tranformed cell `T" on antibiotic agar plate Non-Tranformed cell `C" on antibiotic agar plate Today's experiment - DH5 cells The cells used for this experiment are DH5 cells DH5 is a strain of Escherichia coli bacteria. The DH5 cells are already competent Competent cells are sensitive to stress. Handle them gently. Do not let them warm in your hands. Keep on ice Today's experiment - pUC 18 plasmid pUC 18 plasmid used in this transformation carries an ampicillin resistant gene (encodes -lactamase enzyme). -lactamase enzymes can hydrolyze ampicillin and render it ineffective. PLASMID Today's experiment 2 eppendorf tubes with 25 l of DH5 is provided to you Label each tube "C" for control and the other "T" for transformed cells Tap once gently to make sure the cells are completely thawed Add 5 l pUC19 plasmid to the tube marked `T" Tap 2 times gently to mix the pUC 19 plasmid with the cells Do NOT add anything to the tube marked as "C" Incubate both tubes on ice for 30 mins. Today's experiment Label plates while waiting. All label must be on the bottom edge of plate Place your group and CRN # on all plates Label the two LB agar plates as "C" for control and the other "T" for transformed cells Label the two LB Ampicillin agar -plates as "C" for control and the other "T" for transformed cells Today's experiment
After 20 mins of incubation on ice Place both tubes in 37C water bath for 20 seconds. DO NOT SHAKE Immediately return the tubes to ice for 2 minutes. Add 250 l of LB broth to each. Gently invert the tubes to mix x 2 times. Place 100 l of C on each of the agar plates marked `C" [1 LB and 1 LB/Amp] Spread gently on the surface of the plate using a spreader Place 100 l of T on each of the agar plates marked `T' [1 LB and 1 LB/Amp.] Spread gently on the surface of the plate using a fresh spreader Today's experiment Plate onto four labeled plates as shown below.
LB LB/Amp LB LB/Amp `C' control cells Contamination on plates (mixing spreaders, breathing into plates, touching agar with fingers with give false results : Keep at RT for 10mins for adsorption of the liquid into the LB agar. Invert plates and incubate overnight at 37C Antibiotic resistance will be evaluated in the next lab. `T' transformed cells ...
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- Spring '08