Bacterial Transformation

Plasmids Small circular pieces of extra chromosomal DNA Can be transferred between bacteria via conjugation ( physical contact) Each has one origin of replication and replicates separately from the host DNA Plasmids can be expressed by host bacteria cell In nature plasmids are often lost, unless they contain a gene for antibiotic resistance ( a selective marker). The plasmids that you are working with will use ampicillin as a selective marker to select for bacteria that have actually taken up the plasmid.

Sterile Technique Always open the wrappers of the sterile pipets and loops so that you do not touch the working end. (pipet -from bulbous end, loop from – point end) Never allow the unwrapped circular loop, or the narrow end of the pipet to contact any non sterile object Do not reuse pipets or transfer loops or place them on work station Wash hands thoroughly before and after working with bacteria cultures. Wipe down work bench with 70% ethanol or bleach before and after When transferring things have the loop or bulb ready in one hand. Pick up tube/vial you are transferring to/from and remove cap quickly. Do not drop or put cap down. (A partner might be needed ) Keep fingers away from rim of cap and replace it quickly Do not let pipets or loops touch the sides of the tubes or vials When adding things to and from agar plates, hold lid over the plate to prevent contaminants from falling onto plate surface ( Clam shell)

Helpful Hints When scraping the bacterial cells from the plates do not scrape any agar along with cells. Make sure the cell mass drops in to CaCl2 and doesn’t smear on the side of the tube Resuspend ( Finger vortex) immediately after they are placed in CaCl2. IMPORTANT During heat shock make sure the bacteria being transformed are on ice when carried to the 42 degree water bath. Assign roles – Manager, Data Collector, Assistant, Timer, etc.

What should be included in your lab notebook write up… 1.Title 2.Group Members 3.Background Information (Make sure to define and explain the following concepts: E.coli, transformation, transformation efficiency, plasmids, antibiotics(& resistance) and competent cells.) 4.Purpose – What will happen during the lab (include why transformation is beneficial to bacteria and why it is useful to research scientists 5.Hypothesis for your baseline data and additional variable 6.Drawing of Lab Setup (with labels) 7.Drawings of culture plate results 8.Analysis of Results Please type this up.( Do NOT put the actual student pages in any part of the lab) 9.Conclusion –Answer the Assessment Questions in complete sentences with the corresponding numbers ( Please type this up. Do NOT put the actual student pages in any part of the lab) 10.Further Investigations – What are 3 real world applications of genetic transformations? What are possible ethical, social, or medical issues raised by the manipulation of DNA by biotechnology? Why are these “issues? 11. EXTRA – IN terms of what you have learned about biotechnology, how would you respond to the quote from Michael Crichton’s novel and film Jurassic Park: “Just because science can do something doesn’t mean that it should.” Do NOT include any of the student lab sheets in your formal lab write up!!!

CategoryPoints AwardedComments Title & Group Members (5) Background Info ( 10) Purpose (10) Hypothesis (10) Drawing of Setup (10) Drawing of Results (5) Analysis (14) Conclusion Questions (10) Further Investigation(6) Sub- Total ( /80) Bonus = %+ 10 Grand Total Name ______________________________________Date ______ Block ______ Transformation lab Rubric