Genetics Lab report

BIO 224 L. Hollis-Brown

1 | 11

LAB 7: CHECKING PCR PRODUCTS WITH GEL ELECTROPHORESIS, AND

BACTERIAL TRANSFORMATION

OBJECTIVES 1. Perform a gel electrophoresis on the PCR products of the sgRNA cloning. 2. Interpret the results of the gel electrophoresis. 3. Define genetic transformation. 4. Define bacterial competence. 5. Define antibiotic resistance. 6. Perform a bacterial transformation of Escherichia coli with modified pMZ379. 7. Explain the purpose of the bacterial transformation of E. 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. 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. 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.

BIO 224 L. Hollis-Brown

2 | 11

INTRODUCTION In the previous lab, you cloned the sgRNA genes into the pMZ379 plasmid by PCR. The PCR should have copied the plasmid containing the sgRNA gene, many, many times. In addition, the PCR will have resulted in a linear form of the plasmid. In this week’s lab, you will first run a gel electrophoresis to determine if the plasmid was, in fact, amplified. The gel electrophoresis in this lab will only check to see if the plasmid has been amplified. (You will check in a later lab to see if the sgRNA gene has actually been inserted into the plasmid). Second, you will perform a chemical transformation of bacteria (E. coli) with the modified pMZ379. The PCR you performed last week created only linear copies of the plasmid. If the linear plasmid is taken up by the E. coli during transformation, then the bacteria will ligate the linear plasmid into circular plasmids. In addition, as the E. coli reproduce, they will create many copies of the circularized plasmid. The plasmid must be in circular form in order for it to be taken up and used by the yeast whose genes you will ultimately be modifying.

CHECKING PCR PRODUCTS WITH GEL ELECTROPHORESIS You should be familiar by now with the process of gel electrophoresis. In this week’s lab, we will use a pre-made gel cassette of 1.2% agarose. This cassette fits into a gel dock (instead of a box) that allows the electrical current to travel through the gel. There is no buffer required. Also, the gel has a proprietary DNA stain that allows one to visualize the DNA as it moves through the gel.

Materials

Per Group PCR products from previous lab

1 tube 1X loading dye, 1 mL 1 2-20 μL pipette/with tips (yellow box) 8 sterile microfuge tubes 1 microfuge tube rack Shared by Class 3 13-well FlashGelTM cassettes 1 DNA Ladder premixed with loading dye, 1K-10K 1 Disposal beaker for tubes and tips 1 box Kimwipes 1 bottle DI water

BIO 224 L. Hollis-Brown

3 | 11

Procedure 1. Your instructor will assign students to prepare the FlashGel system using the

following instructions: a) Wear gloves and safety glasses. b) Open the cassette envelope and remove the gel cassette. Be sure not to

touch the gel with bare hands. c) Remove the plastic tape and white well seals. Do not remove the clear side

vent seals. d) Flood the sample wells with DI water, then tip the cassette to allow the excess

water to run off. Blot the gel dry with a Kimwipe. Do not blot the wells as this will remove the liquid from them.

e) Insert the gel cassette into the dock f) Load 4 μL of DNA ladder into the first well of the gel.

2. Each person will load the samples from their gene first (A tubes 1 – 3).

a) Label three sterile microfuge tubes to match your PCR tube contents.

b) Add 5 μL of 1X loading dye to each tube.

c) Add 8 μL of sample from each PCR tube to the appropriate microfuge tube.

d) Slowly pipette the sample five times to mix the dye and the DNA.

3. We will work as a group to load the gels. When it is your group's turn to load your samples into the gel, set the P20 micropipette to 5 μL. Confirm with your lab partner that it is set correctly. Carefully load 5 μL of your sample into the well indicated by your instructor using the following steps:

a) Keep your micropipette steady by stabilizing the shaft of the pipette with the hand not holding the pipette. You can rest your elbow on the table to stabilize your arm.

b) Place the pipette tip just into the top of the well, and slowly release the sample to the first pipette stop. Be careful not to stab through the bottom of the well. The sample will fall to the bottom of the well.

c) Remove the pipette tip from the well, and then release the pipette. Discard the tip.

d) Always use a new pipette tip for each sample.

BIO 224 L. Hollis-Brown

4 | 11

4. Since each gel will be shared, be sure to write down which gel, which lanes, and the contents of the lanes you are assigned, in Table 1 below.

Gel: ______________

Lane # Contents

Table 1. Lanes and their contents in gel of PCR products.

5. When all samples have been loaded into the gel, students will be instructed to: a. plug in the electrodes from the gel box to the power source; b. turn on the power source and the light on the gel box; c. set the voltage to 125, and press Start (set timer for 15 minutes); d. turn off the power source when the ladder has separated, and the smallest

band is almost to the end of the gel; e. place the camera box over the gel and take a picture of the gel on the

computer.

6. Your instructor will print copies of the gels for each student, and also post them on D2L in digital format. You can also take a picture of the gel results. You will need a copy of this image for your final paper.

7. If there is no DNA in your PCR product for your sample, run another gel with the back-up samples from your lab partner using the instructions listed above.

BIO 224 L. Hollis-Brown

5 | 11

Questions Examine your gel image and answer the questions below for both genes. 1. Which lanes showed the presence of DNA?

2. Estimate the size of DNA fragments, just by looking at the ladder and the DNA from your PCR products. What are the estimated sizes of the DNA that appeared? (You do not need to do a standard curve for this.) Be sure to address all fragments of DNA that appear in the gel.

3. Interpret your results. Compare your experimental treatment with the controls. Do you think that you amplified the plasmid by PCR, and why do you think that?

BIO 224 L. Hollis-Brown

6 | 11

TRANSFORMATION OF E. coli WITH pMZ379 Genetic transformation is the uptake and expression of exogenous genetic material by a living cell. Many bacterial and archaeal cells can naturally undergo transformation, by taking up DNA from their surroundings. This process allows them to gain new genes that may provide them with an advantage in certain environments, such as gaining the ability to be resistant to antibiotics, or to break down novel fuel sources. Competence refers to the tendency of bacteria to take up DNA from their environment. Different species or strains of bacteria vary in their competence. Highly competent species will take up DNA relatively easily, while non-competent species will not take up DNA. Even if a bacterial cell is able to take up DNA, it may not necessarily be able to use, or express, the genes that are found on that DNA. Escherichia coli, the bacterial species used in the transformation in this lab, has naturally low to non-existent competence rates (Sinha and Redfield, 2012). Since not all bacteria are naturally competent, competence can be induced in a laboratory setting by artificially making the membranes more permeable to the uptake of DNA. This can be accomplished by heat shock plus chemical means, or by electrical current (a process called electroporation). In this lab, we will use heat shock and chemical means by which to induce competence and transformation in E. coli. The overview of this process is as follows:

• Mix bacteria with exogenous DNA (pMZ379) in a cold environment. • Expose bacteria to ions (these ions neutralize the negative charges on the

phospholipids, so that the negatively charged DNA is not repelled by the membrane.)

• Expose the bacteria to a sudden increase in temperature (this allows large pores to form in the membrane to allow the plasmid to enter.)

• Allow the bacteria to reproduce, and the population to grow, in order to obtain many cells that contain the plasmid.

Question 1. In the last step above, in what kind of environment would you grow the bacteria, in

order to ensure that only the bacteria that have taken up the plasmid will survive, and why?

BIO 224 L. Hollis-Brown

7 | 11

Materials Per Group samples of PCR products from previous lab 4 LB-agar + 75 µg/mL ampicillin plates 1 bottle 95% ethanol, for disinfecting 1 aliquot (200-250 µL ea.) DH5alpha E. coli 1 bottle sterile SOC medium (5 mL) 1 set micropipettors (20, 200, and 1,000 µL) 1 box ea. Large and small, sterile pipette tips 1 foam cooler 1 250 mL beaker for waste 4 sterile flasks, 125 mL 2 Sharpie markers 4 sterile microfuge tubes 2 foam tube racks 2 glass spreading rods 1 gas burner 1 flint Shared by Class 37OC incubator/shaker, with spaces for 20 flasks 1 box ea. Non-latex gloves, small, medium, and large Safety glasses 1 ice bucket and ice 2 rolls masking tape 2 heat blocks Procedure 1. Wear gloves and safety glasses.

2. Disinfect your work station with 95% ethanol.

3. Obtain a foam cooler and fill it with ice.

4. Obtain two LB-agar + 75 µg/mL ampicillin plates.

5. Label the plates with:

• date • group’s initials • first gene name • one plate with “+” (for 1:100) and “0“ (for no plasmid)

BIO 224 L. Hollis-Brown

8 | 11

6. Warm plates in 37OC incubator until ready to use.

7. Keep all materials on ice.

8. Thaw an aliquot of DH5alpha E. coli cells, on ice.

9. Label two microfuge tubes, to match each of the plates from Steps 5 and 6. Be sure that you can tell apart the tubes for the different genes.

10. Place all tubes on ice in one of the foam tube racks. You will move the tubes to the second tube rack, as you add components.

11. Add 50 µL of E. coli to each tube. Move each tube to the second tube rack after you add the bacteria.

12. To the “+” tube for the first gene, add 10 µL plasmid from PCR product Tube 1 (1:100 plasmid DNA -Experimental treatment). Mix gently by tapping with fingers. DO NOT pipette competent cells up and down to mix. Move this tube to the second tube rack.

13. To the “0” tube for the first gene, add 10 µL of solution from PCR product Tube 2 (No plasmid- Control treatment). Mix gently by tapping with fingers. DO NOT pipette competent cells up and down to mix. Move this tube to the second tube rack.

14. Bring your tubes, on ice, to the heat block. Place the tubes in the heat block (43OC) for 1 min. The goal is to transfer the tubes as quickly as possible from one temperature to another. Keep track of which tubes belong to you in the heat block.

15. After 1 min, immediately transfer the tubes back to ice for about 2 min.

16. After 2 min, add 1 mL of SOC medium to each tube. This medium provides ions to facilitate the transformation, and nutrients for the reproducing bacteria. Be sure to use a new pipette tip for each transfer.

17. Obtain 4 sterile flasks, and label them to match your four microfuge tubes. Be sure to include your initials, date, genes, and + or 0.

18. Pipette the solutions from the tubes to the appropriately labeled flasks.

19. Give your flasks to your instructor, who will place them in the shaker at 37°C for 1 hour.

BIO 224 L. Hollis-Brown

9 | 11

Plating Cultures 1. Retrieve your warmed LB+amp plates from the incubator.

2. Plate the cultures from each flask onto the appropriate plate, as follows:

• Light a gas burner and adjust the flame. Use caution around the flame. Do not place the burner where it is near flammable materials, or where a student must reach over it to retrieve materials.

• Squirt a small puddle of ethanol onto the counter, and soak the end of the glass spreader rod in the ethanol. Do not soak the handle in ethanol, or you will get burned.

• While the spreader is still wet, place it in the gas flame and remove it quickly. It will be on fire.

• After the ethanol has burned off, allow the spreader to cool for at least one minute before using it. Do not blow on it or wave it around to cool it down.

• Once the spreader is cool, gently touch it to the agar of the “no plasmid” plate, to transfer any remaining heat to the agar. It is best to start with the “no plasmid” cultures, and then plate the “1:100” cultures. Pour the entire culture of one flask onto the appropriate agar plate, and spread thoroughly with the spreader.

• When finished, place the top on the plate, and reheat the spreader to sterilize it.

• Repeat the above steps for each flask.

3. Allow the plates to sit, agar facing up, until all of the liquid has been absorbed into the agar.

4. Once the culture has been absorbed, stack the plates, agar facing down.

5. Give your plates to your instructor. The plates will be incubated overnight, or longer, at 37OC, then refrigerated.

6. You will check the growth of your bacteria in the next class period.

BIO 224 L. Hollis-Brown

10 | 11

Questions 1. Which plates are the controls and which are the experimental treatments?

2. What do you predict should happen on each plate, if the bacteria were successfully transformed with the plasmid, and why?

3. By the end of this stage in the experiment (transformation), will you be able to determine if each of the following has occurred? Why or why not?

a. The plasmid has been amplified:

b. The sgRNA has been amplified:

c. The E. coli have been transformed with the plasmid:

BIO 224 L. Hollis-Brown

11 | 11

POST-LAB 1. Be sure that gas burners are turned off completely. 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. Empty coolers of ice. 7. Make sure that all materials are clean and returned to your kit. 8. You will turn in this handout for a grade. Be sure you have all questions answered in

the handout.

REFERENCES

Sinha, S. and R.J. Redfield. 2012. Natural DNA uptake by Escherichia coli. PLoS ONE 7(4): e35620. https://doi.org/10.1371/journal.pone.0035620