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Minipreparation of plasmids

Purpose: Minipreparation (miniprep) is done in a laboratory setting in order to extract a plasmid from bacteria. There are several different scales of executing this task, but miniprep is the smallest. The steps of the miniprep are to weaken the cell wall, lyse the cell, precipitate out the lipids and proteins, remove of any chromosomal DNA or RNA that is still present, and then wash the plasmid to make sure that all salts are removed so that only the plasmid remains. This process uses a binding column that is comprised of a silica matrix in order to bind the plasmid DNA so that any other remaining cell fragments can be washed away. This procedure follows the E.Z.N.A. plasmid DNA Mini Kit 1 from the Omega Bio-Tek company.

Vacuum Procedure:

1. A culture was inoculated and grown overnight in a shaker at 37C at 300 rpm.

2. The culture was centrifuged at 10,000 rpm for 1 minute at room temperature

3. The culture median was decanted being careful not to discard any of the cell pellet.

4. 250 µL of Solution I was added and mixed thoroughly and resuspend the pellet. *Note: Solution I is used in order to weaken the cell wall.

5. After the pellet has been resuspended, the contents were added to a microcentrifuge tube.

6. 250 µL of Solution II was added and the microcentrifuge was gently inverted until a clear lysate formed. *Note: Solution II is used in order to lyse the cell wall. Vigorous mixing was avoided to prevent DNA shearing.

7. 350 µL of Solution III was added to the microcentrifuge tube and then it was inverted several times until a white precipitate forms. *Note: Solution III is used to bind all of the proteins and lipids from the cell so that they can be removed.

8. The solution was centrifuged at maximum speed for 10 minutes.

9. The cleared supernatant was transferred to a HiBind DNA Mini Column that was attached to the vacuum.

10. The vacuum was turned on and the liquid was drawn through the mini column.

11. 500 µL of HBC Buffer was added to the column and vacuumed through.

12. 700 µL of DNA wash buffer was added to the column and vacuumed through

13. Step 12 was repeated.

14. The column was added to a collection tube and then centrifuged for 2 minutes on maximum speed to dry the column.

15. The column was transferred to a clean 1.5 mL microcentrifuge tube.

16. 30 µL of sterile deionized water was added to the column and allowed to sit for 1 minute.

17. The microcentrifuge tube with the column inside of it was centrifuged for 1 minute at maximum speed.

18. The column was discarded and the microcentrifuge tube was stored at -20C until the plasmid was needed.

Preparation of Electrocompetent Cells

Purpose: One of the techniques used to incorporate new genetic material into a cell is called electroporation. In this process, electrical pulses create holes in the cell membrane so that genetic material, such as plasmids, is able to permeate the plasma membrane. It is most commonly used in bacterial cells because electroporation is highly efficient at incorporating the new DNA into the bacterial cells that will then be used for cloning. There are special cuvettes that are used for this process that enable the cells to be evenly distributed without air bubbles and for media to be directly added immediately after electroporation occurs so that the cells can recover. Electrocompetent cells are different than normal bacterial cells because they have a weaker cell wall so that electroporation will have a high efficiency.

Procedure:

Part 1: Overnight cell growth

1. 5 mL of LB media were added to a test tube.

2. 5 µL of Streptomycin were added to the same test tube.

3. E. coli cells of the desired strand were used to inoculate the media.

4. The cells were allowed to grow overnight in a shaker at 37C and 300 rpm.

Part 2: Growing Electrocompetent cells

1. 100 mL LB media was added to 2 Erlenmeyer flasks (total 200 mL of media)

2. 100 µL of Streptomycin was added to each of the Erlenmeyer flasks.

3. The optical density (OD) of cells grown overnight culture was measured.

4. The Erlenmeyer flasks were inoculated with enough of the overnight media so that the OD in each Erlenmeyer flask was 0.01.

5. The two flasks were then put in a shaker at 30 C with 300 rpm. *Note: Growing the cells at 30C instead of 37C will alter the growth of the cell membrane

6. Once an OD of 0.5 was reached in the flasks, they were removed from the shaker.

Part 3: Harvesting Electrocompetent cells.

1. While the cells are growing in Part 2, 50 micrcentrifuge tubes were labeled and then set in a box in the -80C freezer.

2. The centrifuge was turned on an cooled to 4C.

3. The contents of the Erlenmeyers flasks were transferred to 4x50mL Falcon Tubes, while making sure the tubes remained chilled.

4. The tubes were put in the pre-chilled centrifuge and spun down at 4500 rpm for 10 minutes.

5. The supernatant was poured out, being careful not to disturb the pellets at the bottom.

6. 25 mL of 10% glycerol was added to each of the tubes and used the gently resuspend the pellet.

7. The contents of the 4 x 25mL Falcon tubes were transferred so that there were 2 X 50 mL Falcon tubes.

8. These 2 tubes were then spun at 4C and 4500 rpm for 10 minutes.

9. Again, the supernatant was poured out and the pellets were resuspended in 25 mL of 10% glycerol.

10. The contents of the 2 x 25mL Falcon tubes were transferred to 1 x 50mL Falcon tube.

11. The tube was spun down again (making sure to balance the centrifuge) at 4C and 4500 rpm for 10 minutes.

12. The supernatant was discarded.

13. This time, the cells were resuspended with 2 mL of 20% glycerol.

14. Quickly, the solution was aliquoted in 55 µL of solution into the pre-chilled centrifuge tubes.

15. The cells were stored in the -80C fridge until they were needed to be used.

Making Agar Plates

Purpose: In order to make antibiotic resistant plates in order to test and make sure that cells have incorporated the desired plasmid.

Procedure:

1. The autoclave was turned on to warm up.

2. 300 mL of distilled water was measured and put in an appropriately sized beaker.

3. 7.5 g of LB miller and 4.5 g of Agar powder were measured and added to the distilled water in the beaker.

4a. The bottle was autoclaved for sterilization purposes.

4b. While the autoclave was running, ~16 Petri dishes were labeled with the date and the type of antibiotic that would be added to the plates.

5. The bottle was removed from the autoclave and then allowed to cool to about 60 C.

6. When the bottle was cooled, 300 µL of the desired antibiotic was added to the solution.

7. The bottle was swirled to ensure that the antibiotic was mixed equally in the solution.

8. Approximately 50 mL of the solution was distributed to each of the Petri dishes, under sterile conditions, and allowed to cool.

9. Once the agar had solidified, they were placed lid side down and stored in the refrigerator. *Note: Lid side down is important so that any condensation does not collect on the agar. This could affect the quality of the plates.

Transformation

Purpose: This procedure is done in order to incorporate new genetic material into a cell. This lab uses transformation on bacterial cells to insert plasmids. Once the desired plasmids have been constructed and isolated, they are inserted in to the cells by electroporation or heat shock. Once these cells have the new genetic material introduced, they are allowed to grow and recover in a shaker and then are plated so that cells without the incorporated antibiotic resistance and the new gene will die.

Procedure:

1.A 50 µL aliquot of electrocompetent cells were allowed to thaw on ice.

2. An electroporation cuvette was put on ice to chill.

3. 2-3µL of the desired plasmid was added to the electrocompetent cells and mixed gently by pipetting.

4. The cuvette was wiped and then placed in the electroporator. *Note: For E. Coli, the electroporator should be set for 2500 V

5. The time constant was recorded (should be between 4.6 and 5.6)

6. 600 µL of SOC was added directly to the cuvette and mixed with the cells.

7. The cuvette was incubated at 37 C at various times depending on the antibiotic resistance marker:

Ampicillin – 15 min

Chloramphenicol – 30 min

Kanamycin/Streptomycin – 45 minutes

8. After the incubation period, the cells were added to the pre-made agar plate and spread using a sterilized spreader.

9. The plate was put in the 37 C oven for the cells to grow overnight.

Polymerase Chain Reaction

Purpose: Polymerase Chain Reaction (PCR) is a laboratory technique that is used to help amplify specific sections of DNA. These strands of DNA are then able to be analyzed and put into new cells to see how their behavior is affected. This technique is mostly used when a cell is being designed that will use genes from multiple sources. DNA is able to replicate on its own, but it takes several hours to occur. PCR is able to optimize the temperature and process that is needed for replication to happen so that instead of taking several hours in order for one copy to be made, thousands can be made in the same amount of time. Each reaction follows the same three steps; denaturation, annealing, and extension. This procedure is the suggested protocol by New England BioLabs Inc.

Procedure:

1. A bucket of ice was gathered to ensure that all materials remained cold.

2. The following components (total 50µL) were added to a PCR tube, from largest quantity to smallest:

Nuclease-free water - 37 µL