Bacterial Transformation LAB - Mr. G's AP Bio

Bacterial Transformation LAB - Mr. G's AP Bio

What did we try to show? Bacterial Transformation LAB When I come around please flip notebook to the procedure you completed before starting the lab yesterday. 20 points. Can recover lost points by writing a good practice hypothesis: Null/alternative OR If/then statement 5. Read the lab safety information and write the complete procedure (pp. S103-S105) in your notebook. THIS MUST BE DONE BEFORE YOU CAN PROCEED WITH THE LAB.

3. Write a simple hypothesis by comparing your sample with that of a partners, or design what to sample and test. 6. Make a hypothesis. Bacterial Transformation LAB Form a hypothesis by following the general outline given at the beginning of the year: Hypotheses (write all possible outcomes of experiment) H0 : the null hypothesis; the hypothesis of no difference; e.g. the test group results were not different from the control group results (A=B) H1 or HA : the alternate hypothesis; the hypothesis of difference; the test group results were significantly different than the control group (AB) There can be more than one alternate

hypothesis and they can be directional (<,>,,, ) Identify the: Independent Variable (I.V.) = the one that you can control the change, this is the x-variable on a graph Dependent Variable (D.V.) = the one that you measure the change, this is the y-variable on a graph Bacterial Transformation LAB What were you testing? Think about what was in the plate. - LB LB What was expected to happen?

What does growth mean? What does no growth mean? Why the different plates? What will growth or no growth in the plates tell you? + LB- amp + LB-amp Bacterial Transformation LAB So in your hypothesis include the following: 1. What is the null hypothesis? a. If the plasmid was not taken up, we expect to find growth

b. What does the bacteria without the plasmid tell us? (i.e. Why a control group?) 2. What is the alternative hypothesis? a. If the plasmid was taken up we expect to find growth b. What does the LB-only plate tell us? (i.e. How is this a control?) 3. What are your variables? Bacterial Transformation LAB Answer Analyzing Results questions p. S105 But put before the Analyzing Results section. Analyzing Results

Think about these questions before collecting data and analyzing your results. Be sure to record your answers in your laboratory notebook. 1. On which of the plates would you expect to find bacteria most like the original nontransformed E. coli colonies you initially observed? Why? 2. If there are any genetically transformed bacterial cells, on which plate(s) would they most likely be located? Again, why? 3. Which plates should be compared to determine if any genetic transformation has occurred? Why? 4. What barriers might hinder the acquisition of plasmids? 5. How can the procedures described above (addition of CI2 and heat shocking) help facilitate the introduction of plasmids into the E. coli cells? Bacterial Transformation LAB Answer the next set of questions (p.S106)in essay form in your

conclusion. 1. Do your results support your original predictions about the + plasmid transformed E. coli cells versus - plasmid nontransformed cells? 2. Which of the traits that you originally observed for E. coli did not seem to become altered? Which traits seem now to be significantly different after performing the transformation procedure? 3. What evidence suggests that the changes were due to the transformation procedures you performed? 4. What advantage would there be for an organism to be able to turn on or off particular genes in response to certain conditions? 5. Was your attempt at performing a genetic transformation successful? If so, how successful? Bacterial Transformation LAB

Complete a data table in the results section of the write-up by looking at your plates Bacteria Plasmid present agar E. coli E. coli E. coli E. coli + + LB LB-amp LB LB-amp

growth Number of individual colonies Bacterial Transformation LAB Transformation efficiency = Calculations DNA in g =g = We will use class data: (concentration of DNA of g =g/L) x g =L) x 7 colonies total (volume of DNA in g =L) Multiply your groups DNA mass () by 5 ) by 5

because there were 5 groups What was the volume of the DNA solution you put on the plate? Bacterial Transformation LAB Remember 5 groups: 1. Calculate the total number of transformed cells. n=7 Calculate the amount (mass) of plasmid DNA (pAMP) in g per 1 L of g per 1 g per 1 L of L of solution. pAMP= (0.005g =g/L) x g =L) x 10g =L pAMP = 0.05 g per 1 L of g x5 = 0.25 g per 1 L of g

Bacterial Transformation LAB Fraction of DNA used = Volume spread on the LB/L) x amp plate (g =L) Total sample volume in test tube (g =L) Fraction DNA = Remember 5 groups: x = 0.2

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