Genetic Engineering:
Directions for Laboratory Period 3

This Week's Objectives | This Week's Schedule | Materials | Procedure | Assignment | Acknowledgments | Genetic Engineering Table of Contents

This Week's Objectives

Last week you constructed recombinant plasmids by ligating together two purified DNA fragments and then transforming the mixture into competent E. coli cells. This week we will screen these transformed colonies to determine if any contain the desired recombinant plasmid.

Specifically, we will...

• Isolate plasmid DNA from transformed colonies.
• Analyze their size by agarose gel electrophoresis.

This Week's Schedule

The Day Before:

1. Inoculate cultures with transformed bacteria (from last week)
2. Incubate the cultures overnight.

On The Scheduled Lab Day:

1. Harvest the bacterial cultures and prepare plasmid DNA using the alkaline lysis procedure (see "The Alkaline Lysis Procedure for Preparing Plasmid DNA") (~1 hour).
2. Compare the size of these plasmids to the original plasmids by agarose gel electrophoresis (~1 hour).

Materials

The following equipment and supplies will be available:

• Sterile culture tubes
• SOB broth + ampicillin
• SOB agar plates + ampicillin
• Glucose/EDTA/Tris (GET) solution (50 mM glucose, 25 mM Tris-HCl, pH 8.0, 10 mM EDTA)
• SDS/NaOH (1% SDS, 0.2N NaOH
• 3M potassium acetate (KOAc, pH 4.8)
• isopropanol
• ethanol
• T.E. (10 mM Tris-HCl, pH 8.0, 1 mM EDTA)
• Deionized H2O
• 6X Loading dyes
• Sterile toothpicks
• 37°C Shaker incubator
• P-20 pipetman
• P-200 pipetman
• 1 ml pipets
• Sterile yellow pipet tips
• Sterile 1.5 ml tubes
• Microcentrifuge
• Vortex Mixer
• Gel boxes with lids
• Gel electrophoresis buffer (1x TAE)
• Electrical power supplies
• Gloves
• Weighing balance
• Agarose
• Ethidium bromide staining solution (@ 0.5 µg/ml)
• Ultraviolet transilluminator
• Ultraviolet-shielding safety goggles
• Film
• Film holder and hood
• Waste containers for solid and liquid materials contaminated with ethidium bromide

Procedure (To be performed with a partner)

 

Inoculate cultures (the day before)

1. Obtain and label six tubes of sterile SOB + ampicillin broth and one SOB + ampicillin plate. Also retrieve the plate of bacteria transformed with your ligation mixture (not the control plate).
2. Using a sterile toothpick, sterilely transfer bacteria from a single colony to a new plate (as a half inch streak) first and then place the entire toothpick into an appropriately labelled tube. Repeat this procedure for a total of six transformants.
3. Transfer the six inoculated tubes to the 37°C shaker (in Mateer 308). Shake overnight at 37°C, 250 rpm.

 

Isolate plasmid DNA

1. The next day, label six 1.5 ml tubes.
2. After retrieving your overnight cultures, shake each culture tube to resuspend the E. coli cells. Transfer 1.5 ml of each culture to a separate, labelled 1.5 ml tube.
3. Close the caps of each tube, and place them in a balanced configuration in the microfuge rotor. Spin for 1 minute to pellet the cells.
4. Discard the supernatant from each tube into the original culture tubes. Completely drain each 1.5 ml tube by inverting it on a paper towel. Be sure no droplets remain.
5. Add 100 µl of the GET solution to each tube and resuspend each pellet by vortexing. Verify that the suspension is homogeneous and that no visible clumps of cells remain.
6. Add 200 µl of SDS/NaOH to each tube. Mix the solutions by inverting the tubes five times. Do not vortex!
7. Incubate the tubes on ice for 5 minutes. The suspension will become relatively clear.
8. Add 150 µl of ice-cold 3M KOAc (pH5.2) to each tube. Mix the solutions by inverting the tubes five times. A white precipitate should appear immediately.
9. Incubate the tubes on ice for 5 minutes.
10. Spin the tubes for 5 minutes to pellet the precipitate.
11. Using a 1 ml pipet, transfer 400 µl of the supernatant from each tube into clean 1.5 ml tubes. Avoid pipetting the precipitate. Discard the old tubes containing the precipitate.
    STOP! Coordinate the next step with other class members so that you can quickly proceed to the subsequent step without delay.
12. Add 400 µl of isopropanol to each tube of supernatant. Mix vigorously by rapidly inverting the tubes five times. Stand at room temperature for only 2 minutes.
    Isopropanol preferentially precipitates nucleic acids rapidly; however, proteins remaining in the supernatant also begin to precipitate with time.
13. Spin the tubes for 5 minutes to pellet the nucleic acids.
    Align the tubes in the rotor so that the cap hinges point outward. The nucleic acid residue, visible or not, will collect under the hinge during centrifugation.
14. Pour off the supernatant from both tubes. Be careful not to disturb the nucleic acid pellets. Invert tubes, and tap gently on the surface of a clean paper towel to drain thoroughly.
15. Add 200 µl of 100% ethanol to each tube, close the caps, and flick the tubes several times to wash each pellet.
16. Spin the tubes for 2-3 minutes.
17. Pour off the supernatant from all tubes. Be careful not to disturb the nucleic acid pellets. Invert each tube, and tap gently on the surface of a clean paper towel to drain thoroughly.
18. Air dry the nucleic acid pellets for 10 minutes.
    To save time, you may want to prepare you agarose gel now (see "Perform agarose gel electrophoresis" below)
19. At the end of the drying period, hold each tube up to light to check that no ethanol droplets remain. If ethanol is still evaporating, an alcohol odor can be detected by carefully sniffing the mouth of the tube. All ethanol must be evaporated before proceeding.
20. Add 15 µl of TE to each tube and resuspend the pellets by repeated pipetting. Check that all DNA is dissolved and that no particles remain in the tip or on the side of the tube.

 

Perform agarose gel electrophoresis

1. Prepare an 0.8 % agarose gel in 0.5X TAE (as described in Laboratory #1; briefly, dissolve 0.12 g of agarose in 15 ml TAE.)
2. Mix 2 µl of each plasmid prep DNA with 6 µl dH2O and 2 µl 6X dye.
3. Load each of the six samples into separate wells of the gel. Also, load 10 µl samples of the original plasmids (uncut).
4. Run the gel at 100 volts for 1 &endash; 2 hours (as described in Laboratory #1).
5. When done, stain the gel for ten minutes in ethidium bromide, visualize with ultraviolet light and photograph (as described in Laboratory #1).

Assignment

Due One Week Later

Either jointly or individually prepare a complete lab report following the guidelines described in Pechenik's A Short Guide to Writing about Biology, as summarized in the lab manual section entitled "Brief Descriptions of the Sections of a Laboratory Report".

Acknowledgments

The following sources were used in the preparation of this week's laboratory directions: DNA Science: A First Course in Recombinant DNA Technology by D.A. Micklos and G.A. Freyer (1990); Cold Spring Harbor Laboratory Press, Cold Spring Harbor. Molecular Cloning: A Laboratory Manual by J. Sambrook, E.F. Fritsch, and T. Maniatis (1989); Cold Spring Harbor Laboratory Press, Cold Spring Harbor

Genetic Engineering (Molecular Genetic Analysis)

• Overview
• Directions for Laboratory Period 1
• How to Interpret the Results of the Restriction Enzyme Analysis
• Directions for Laboratory Period 2
• The Alkaline Lysis Procedure for Preparing Plasmid DNA
• Directions for Laboratory Period 3

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