Genetics Lab report

BIO 224 L. Hollis-Brown

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LAB 8: TRANFORMATION RESULTS AND PLASMID RECOVERY

OBJECTIVES 1. Record and interpret results from a bacterial transformation. 2. Define DNA isolation. 3. Isolate plasmid DNA from bacterial cells. 4. Explain the purpose of the isolation of pMZ379 from Escherichia coli in the context of

CRISPR/Cas gene editing in fission yeast. BEFORE LAB 1. Read the lab handout in its entirety. If you do not prepare adequately for lab,

you will not be able to complete the lab in the time allotted. 2. Watch the video on the plasmid recovery protocol (also linked on D2L.) 3. Complete the pre-lab quiz before lab.

LAB SAFETY 1. Wear gloves and safety glasses throughout the procedure. 2. Clean your workstation before and after the procedure. 3. Wear closed-toed shoes and secure loose hair and clothing. 4. Dispose of reagents and used materials in labeled containers only. 5. Dispose of bacterial culture plates in marked biohazard containers only. 6. Use caution around open flames.

GENERAL NOTES ON LAB 1. Be sure to ask questions if you are unsure of any instructions.

2. Take careful notes as you do the lab. You will need these notes to write your final paper at the end of the semester.

3. You will turn in this lab handout for a grade. Be sure you have all questions answered in the handout.

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INTRODUCTION In the previous lab, you transformed Escherichia coli with the plasmid pMZ379 that had the sgRNA gene inserted into it. This was done in order to change the linear form of the plasmid, created by the PCR, into a circular form. The E. coli not only circularized the plasmid, but also created many copies as the bacteria reproduced. In this lab, you will: 1.) record and interpret the results from your bacterial transformation experiment from last week; and, 2.) recover, or isolate, the circularized, modified plasmid from the bacterial cells. The technique of DNA isolation separates out the DNA from all of the other cellular components. Recall that DNA is one type of biological macromolecule, made of nucleotide monomers. DNA must be separated from all of the other cellular components, such as RNA, lipids, carbohydrates, proteins, and salts, in order to obtain a solution of pure plasmid. The procedure in this week’s lab is based on a proprietary protocol from the Qiagen QIAprep® Spin Miniprep Kit. From the QIAprep® handbook (QIAgen, 2015):

The QIAprep miniprep procedure is based on alkaline lysis of bacterial cells followed by adsorption of DNA onto silica in the presence of high salt.

The procedure consists of three basic steps: • preparation and clearing of a bacterial lysate; • adsorption of DNA onto the QIAprep membrane; • washing and elution of plasmid DNA.

Next week, you will determine if the purified plasmid contains the sgRNA gene through a restriction enzyme digest. In the subsequent labs, you will transform the fission yeast with the modified, circularized plasmid.

Questions 1. What is the general purpose of DNA isolation?

2. What is the specific purpose of this week’s lab?

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CHECKING TRANSFORMATION RESULTS Last week in lab, you plated E. coli that you hoped had been transformed with pMZ379 containing the sgRNA gene and Cas9 gene. You will record and interpret the results of this transformation.

Examine your culture plates and record your results in Table 1 below.

Plate/Contents Predicted Results (growth or no growth?)

Observed Results (growth or no growth?)

Table 1. Transformation Results

Questions

1. For your gene, were the bacteria transformed with the plasmid? Explain how you know. (You must reference your controls in your explanation.)

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RECOVERY OF pMZ379 FROM E. coli Follow the procedure exactly as it is written. When you are finished, you should have a small quantity of pure plasmid containing each sgRNA gene.

Materials

Per Group overnight cultures of transformed E. coli from previous lab, in 125 mL flasks

1 set 2-20, 10-100, and 100-1000 μL pipettors 1 each box of sterile large and small pipette tips 1 bag sterile microfuge tubes 1 plastic microfuge tube rack 2 QIAprep® spin columns 2 fine Sharpies 1 each aliquot of following:

• 600 µL Buffer P1 (LyseBlue and RNaseA already added), stored at 4OC

• 600 µL Buffer P2 • 750 µL Buffer N3 • 1.5 mL Buffer PE (ethanol already added) • 125 µL Elution Buffer (EB)

1 250 mL waste beaker for tubes and tips 1 bottle 95% ethanol for disinfecting Shared by Class 2 microcentrifuges biohazard waste containers for bacterial plates 1 ice bucket with ice 1 box ea. non-latex gloves, small, medium, large safety glasses Procedure 1. All steps may be carried out at room temperature (15-25OC). The Buffer P1 is

stored on ice at the front of the room until you are ready to use it.

2. Wear gloves and safety glasses, and secure loose clothing and hair.

3. Clean your workstation, pipettors, and writing utensils with ethanol.

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4. Obtain the overnight culture flasks of bacteria that you transformed. Recall that, in the previous lab, you transformed the bacteria in liquid culture, and then transferred the bacteria to agar plates containing ampicillin. A colony from each of your plates was then transferred to another liquid culture containing ampicillin and grown overnight.

5. Obtain two sterile microfuge tubes, and label them with the name of your gene and your initials.

6. Obtain two spin columns, and label them with the name of your gene and your initials. Be careful not to touch the inside of the spin column.

7. Use the P1000 pipettor to transfer 1 mL of each culture from your labeled flasks to one of each of the appropriately labeled sterile microfuge tubes. You will fill the first tube with the culture from the first flask, and then you will fill the second tube with the culture from the second flask (i.e., one tube for each flask).

8. Place your microfuge tubes in the microcentrifuge, as instructed below. Share the microfuge with other groups, whenever possible. It is imperative that you follow these instructions, or you may damage the centrifuge.

• The placement of the tubes must be balanced. In other words, there must be equal spaces between the tubes or groups of tubes.

• Place the hinges of the tubes facing outward. • When all tubes are loaded, place the small plastic lid firmly on the top. • Close the lid of the centrifuge. • Set the speed (rpm) and time (min) (see details in the protocol). • Press the start button. • Wait until the centrifuge has stopped completely before removing tubes.

9. Centrifuge at 13,000 rpm for 3 min. When finished centrifuging, there should be an

obvious, cream-colored pellet of bacterial cells stuck to the bottom of each tube. This step concentrates the cells.

10. Perform all of the next steps for both of your tubes.

11. Pour out the supernatant into your waste beaker, without disturbing the pellet of cells. Do not tap or shake the tube. It is okay if there is still a little liquid left in the tube.

12. Resuspend the pelleted bacterial cells in 250 μL Buffer P1 by gently pipetting the cells up and down into the solution. This buffer suspends the cells in solution, or solubilizes them, and also degrades the RNA.

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13. Add 250 μL Buffer P2 and mix thoroughly by inverting the tube 4–6 times until the solution turns from cloudy to blue. This step lyses the bacterial cells, releasing their components. It allows the DNA to remain in solution while the other components precipitate out of solution. (Buffer P1 also contains a color indicator that shows correct mixing of the buffers.) Do not allow the lysis reaction to proceed for more than 5 min.

14. Add 350 μL Buffer N3 and mix immediately and thoroughly by inverting the tube 4– 6 times. The solution will turn colorless. This step neutralizes the solution and allows the DNA in the solution to bind to the “membrane” in the spin column in Step 16.

15. Centrifuge for 10 min at 13,000 rpm. Since this step takes the longest, you should share the centrifuge with other groups. This step allows the cellular debris to precipitate out of solution into a pellet while the plasmid DNA remains in the solution.

16. After centrifuging, the DNA is in the supernatant, or the liquid at the top of the tube. Remove 800 μL of supernatant from the tube, without disturbing the pellet at the bottom. Transfer the supernatant to the appropriately labeled QIAprep 2.0 spin column.

17. Place the spin columns in the centrifuge (no caps are necessary for the tubes), and centrifuge for 60 sec.

18. Pull out the spin column from its tube, and discard the flow-through by pouring it into your waste beaker. Place the column back in the tube. At this point, the DNA is adsorbed to the membrane in the spin column.

19. Wash the spin column by adding 750 μL of Buffer PE to the column. This step removes impurities (such as proteins and salts) that are still on the membrane.

20. Centrifuge for 60 sec.

21. Remove the spin column from its tube, and discard the flow-through.

22. Centrifuge for 1 min to remove residual wash buffer.

23. Remove the spin column from its tube and transfer it to an appropriately labeled, sterile microfuge tube. Discard the tube that came with the spin column.

24. Add 50 μl Buffer EB to the center of the spin column, let stand for 5 min, and then centrifuge for 1 min. (Turn the open caps toward the inside of the centrifuge). This step elutes the DNA, or removes it from the spin column and washes it into the solution into the microfuge tube.

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25. The pure plasmid DNA is now in the solution in the microfuge tube. Remove the spin column and discard it.

26. Cap the microfuge tube. Make sure it is labeled with your group’s initials, “plasmid”, and the correct gene.

27. Measure the concentration of the DNA using the Nanodrop.

a. Mix the contents of the tube by flicking the tube then briefly centrifuging the tube.

b. Add 1µL of the DNA solution to the Nanodrop platform then lower the arm.

c. Note the concentration of the tube on the tube itself as well in the blank below. Concentration is listed in µg/µL or ng/µL

d. Repeat the procedure for the second tube.

Tube 1 concentration: _____________ Tube 2 concentration: _____________

28. The purified plasmid will be stored at -20OC until needed.

Questions 1. By the end of this protocol, you should have a solution of purified plasmid. Identify

the structure and specific features that are found on this plasmid.

2. Describe two reasonable errors that might have occurred during your procedure. Do not make up an answer. Try to answer honestly about what errors might have happened.

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POST-LAB 1. Dispose of all culture plates in biohazard containers. 2. Be sure all tips and tubes are disposed of in the waste beakers. 3. Wipe down your workstation with ethanol or Lysol. 4. Throw away all gloves and paper towels. 5. Wash your hands. 6. Make sure that all materials are clean and returned to your kit. 7. You will turn in this handout for a grade. Be sure you have all questions answered in

the handout.

REFERENCES QIAgen, 2015. QIAprep Miniprep Handbook.