18.3: Lab Report

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Exercise A

Transformation Day 2: Results, Analysis, and Critical Thinking

After the plates have been incubated for 24-48 hours, observe for growth. Which plates did E. coli grow on? Indicate growth by circling the plates below:

LB- LB+ LB/Amp- LB/Amp+

A1. What was the purpose of the “LB-“ plate?

A2. What was the purpose of the LB/Amp- plate?

A3. Why was there no growth observed on the LB/Amp- plate?

A4. Did your results support your hypothesis? Yes / No (circle one)

A5. Based on your results, would it be possible for ampicillin resistance to be transferred (horizontally) from one bacterium to another?

Explain how this might occur:

Transformation efficiency is expressed as the number of antibiotic-resistant colonies per microgram of pAMP. On which plate will only the antibiotic-resistant colonies grow? ______________________ Use this plate to determine transformation efficiency below:

A6. Determine the total amount of pAMP you added to the “+” microtube: ________µg

(Note: one loopful = 10 µL, and the concentration of the pAMP stock solution was 0.2 µg / µL)

A7. Determine the total concentration of pAMP (in µg / µL) in the total suspension of cells plus Luria broth used for recovery (250 µL CaCl₂ + 10 µL pAMP + 250 µL Luria broth): ___________ µg / µL

A8. Total amount of pAMP in the 100 µL spread on the “+” plates: ______ µg pAMP/ 100 µL

A9. Count the number of colonies on the plate: _____________ colonies. (If there are too many colonies to count, divide the plate into quarters or eighths, count one section, and multiply by the number of sections.)

A10. Divide the number of colonies by the amount of pAMP in the 100 µL of cell suspension spread on the plate (step 8) to find the colonies/µg pAMP (scientific notation): __________ colonies / µg pAMP. This is the transformation efficiency for your group.

A11. Record the transformation efficiencies for the other groups in our lab:

A12. Find the average transformation efficiency for our class: _________________ colonies / µg pAMP

Exercise B

B1. What is the name of the enzyme that connects, or seals, the DNA fragments together?

B2. What do you notice about the 3’ and 5’ ends of the target gene and the plasmid cut site?