BioBuilding: Synthetic Biology for Teachers: Lab 4: Difference between revisions
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# Label 2 small eppendorf tubes either "4-1" or "4-2". | # Label 2 small eppendorf tubes either "4-1" or "4-2". | ||
# Pipet 200 ul of "TB" transformation solution into each eppendorf and then place the tubes on ice. | # Pipet 200 ul of "TB" transformation solution into each eppendorf and then place the tubes on ice. | ||
# Use a sterile wooden dowel to scrape up | # Use a sterile wooden dowel to scrape up one entire patch of cells (NOT including the agar that they're growing on!) labeled "4-1," and then swirl the cells into its tube of cold "TB." A small bit of agar can get transferred without consequence to your experiment, but remember you're trying to move the cells to the "TB," not the media they're growing on. If you have a vortex, you can resuspend the cells by vortexing for one minute. If no vortex is available, gently flick and invert the eppendorf tube. | ||
# Repeat, using a different sterile wooden dowel to scrape up the patch of cells labeled "4-2." Vortex briefly if possible. It's OK for some clumps of cells to remain in this solution. | # Repeat, using a different sterile wooden dowel to scrape up the patch of cells labeled "4-2." Vortex briefly if possible. It's OK for some clumps of cells to remain in this solution. | ||
# Keep these competent cells on ice while you prepare the DNA for transformation. | # Keep these competent cells on ice while you prepare the DNA for transformation. | ||
====Part 2: Transforming Strains 4-1 and 4-2 with pPRL and pGRN==== | ====Part 2: Transforming Strains 4-1 and 4-2 with pPRL and pGRN==== | ||
The cells you've prepared will be enough to complete a total of 6 transformations. You will transform the purple-color generator into each strain, and also the green-color generator into each strain. You will also use the last bit of competent cells as negative controls for the transformation. A video of this procedure is [http://youtu.be/ayvElUIc0pg here.] | The cells you've prepared will be enough to complete a total of 6 transformations. You will transform the purple-color generator into each strain, and also the green-color generator into each strain. You will also use the last bit of competent cells as negative controls for the transformation. A video of this procedure is [http://youtu.be/ayvElUIc0pg here.] | ||
[[Image:Note mini.png]]''<font color = red> TEACHERS: It is important that the students correctly label each tube and plate. </font color> <br> | [[Image:Note mini.png]]''<font color = red> TEACHERS: It is important that the students correctly label each tube and plate. </font color> <br> | ||
# Retrieve 2 aliquots (in eppendorf tubes) of each plasmid for a total of 4 samples (2x pPRL, 2x pGRN). Each aliquot has 5 ul of DNA in it. The DNA is at a concentration of 0.04 ug/ul. You will need these values when you calculate the transformation efficiency at the end of this experiment. | |||
#Label one the pPRL tubes 4-1 and the other 4-1 (be sure that the pPRL label is readable)! | |||
#Label one of the pPRL tubes 4-1 and the other 4-1 (be sure that the pPRL label is readable)! | |||
# Retrieve 2 aliquots of each plasmid for a total of 4 samples (2x pPRL, 2x pGRN). Each aliquot has 5 ul of DNA in it. The DNA is at a concentration of 0.04 ug/ul. You will need these values when you calculate the transformation efficiency at the end of this experiment. | |||
# Pipet 5 ul of each DNA into the appropriate tube, i.e. the purple-color generator (pPRL) into a tube for transforming strain 4-1 and another for 4-2, then the green-color generator (pGRN) into the remaining two tubes. Store the tubes on ice. | # Pipet 5 ul of each DNA into the appropriate tube, i.e. the purple-color generator (pPRL) into a tube for transforming strain 4-1 and another for 4-2, then the green-color generator (pGRN) into the remaining two tubes. Store the tubes on ice. | ||
# Flick the tube with the competent 4-1 strain and then pipet 75 ul into the tube labeled "pPRL, 4-1" and an additional 75 ul into the tube labeled "pGRN, 4-1." Flick to mix the tubes and return them to the ice. Save the remaining small volume of the 4-1 strain on ice. | # Flick the tube with the competent 4-1 strain and then pipet 75 ul into the tube labeled "pPRL, 4-1" and an additional 75 ul into the tube labeled "pGRN, 4-1." Flick to mix the tubes and return them to the ice. Save the remaining small volume of the 4-1 strain on ice. |
Revision as of 08:36, 8 August 2011
Eau That Smell Lab notes |
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Lab 4: What a Colorful WorldTeacher ConsiderationsThis lab provides a streamlined and effective alternative to more common transformation protocols. The engineering concept of chassis gives this lab a context that takes it beyond a simple exploration of the technique. Using the protocol presented below, we generally observe strain 4-1 produces large, light green colonies and dark purple colonies. The 4-2 strain usually produces dark, small green colonies and no purple colonies. We generally observe transformation efficiencies around 1*10^3 colonies/microgram of DNA. However, variations on the protocol, such as incubation at room temperature, may produce different results. We encourage you to experiment and report your results back to us. To achieve high transformation efficiency and clear color differences, it is important that students be precise when conducting the transformation protocol. For instance, a water bath in excess of 42oC or leaving the cells in the bath for more than 90 seconds may damage the cells and adversely affect the transformation efficiency. If time allows, it may be worth having the students practice the procedure using water samples the day before the actual lab. Needed MaterialsTeacher Provides
Kit Provides
WorkflowAdvance preparation TEACHERS: You will be receiving two strains of bacteria to transform. These strains will consist of colonies of each strain on a petri dish. To obtain sufficient amounts of bacteria for a class to conduct transformation, you will need to replate these colonies as patches. Each patch will provide sufficient bacteria for 1 lab group and up to 6 patches will fit comfortably on one plate. This process can be done by the student or the teacher up to a week prior to the transformation. You may wish to conduct this procedure yourself. However, if time allows, the students will enjoy learning these microbiological techniques.
TEACHERS: On the day that the students will transform the cells, you will need to complete the transformation buffer by adding DMSO to a final concentration of 10%. Each group will need at least 0.4 ml of TB+DMSO. We recommend making 500μL to account for pipet variations. To make a TB+DMSO solution, add 50μL of DMSO to 450 μL of TB per each group, flick to mix and chill in fridge or on ice. Annotated Laboratory Procedure TEACHERS: These are the same instructions as included on the student site but with special notes for the teacher. Part 1: Preparing Strain 4-1 and 4-2 for transformationNeither of these E. coli strains will take up DNA from the environment until they are treated with a salt solution that makes their outer membrane slightly porous. The cells will become "competent" for transformation (i.e. ready to bring DNA that's external to the cell into the cytoplasm where the DNA code can be expressed). The cells will also become fragile. Keep the cells cold and don't pipet them roughly once you have swirled them into the "TB" salt solution. TEACHERS: You should emphasize that this procedure can be tough on the cells. The students should be gentle and work quickly.
Part 2: Transforming Strains 4-1 and 4-2 with pPRL and pGRNThe cells you've prepared will be enough to complete a total of 6 transformations. You will transform the purple-color generator into each strain, and also the green-color generator into each strain. You will also use the last bit of competent cells as negative controls for the transformation. A video of this procedure is here.
TEACHERS: It is important that the students correctly label each tube and plate.
TEACHERS: This videoillustrates the use of a sterilized spreader as well as sterile beads. Packaged sterile spreaders could also be used and do not have to be sterilized with alcohol before spreading the cells, but do have to be changed between samples. Next dayIn your lab notebook, you will need to construct a data table as shown below. These may be provided.
TEACHERS: Clean-up instructions. Provide containers at each work stations for student biological waste such as pipet tips, eppendorf tubes, spreaders, innoculating loops, and plates. Be sure to follow hazardous waste procedures as set forth by your school or municipality. Generally, it is safe to soak the material in each container with a 10% bleach solution for 2 hours. Materials can then be discarded into the regular trash. You can find more information about microbiology lab safety here CalculationsHere is a sample calculation for transformation efficiency
Calculation:
Sample Data
AssessmentLab Report RubricLab Report ScoreSheetSurveyTo help us improve the labs, you can
Thanks! Variations to try
FeedbackWe're always looking to hear back from you if you've thought about this unit, tried it, or stumbled across it and want to know more. Please email us through BioBuilder, info AT biobuilder DOT org.
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