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Assignments Due

• A Genetic Switch, 3rd Edition, Mark Ptashne: Chapters 1-3 (pp. 11-65), Appendix 1 (pp. 141-147)

Lecture Topics

• Course introduction
• A genetic switch: Joff Silberg (Invited Speaker)

Overview of Experiment

Background

• Sterile Technique
• Bacterial Transformation and Selection
• Agarose Gel Electrophoresis of DNA

Notes on Molecular Biological Procedures

General Guidelines
1. Maintain a clean work area
2. Use a fresh pipet tip for every transfer (tips should be DNase/RNase free)
3. Wear gloves to prevent nuclease contamination (and to protect yourself!)
4. Sterilize solids and liquids by autoclaving 20 minutes at 121°C at 15 psi

Pipetting Small Volumes
1. Before beginning the procedure, thaw all frozen reagents and mix well
2. Pulse spin ALL tubes of aliquots to bring the liquid to the bottom of the tube -- in the microcentrifuge hold the "SHORT" key for about 5-10 seconds
   (Capless 1.5 ml vials serve as holders for 0.2 and 0.5 ml tubes in the microcentrifuge rotors)
3. Touch only the very tip to the surface of the solution (i.e., do NOT submerge the pipet tip into the solution)
4. Most enzyme stocks are in 50% glycerol; these solutions are quite viscous and liquid will stick to the outside of the pipet tip so touch only the surface

Centrifugation
1. DO NOT PUT TAPE ON TUBES!
2. ALWAYS balance the load in the centrifuge
3. Capless 1.5 ml vials serve as holders for 0.2 and 0.5 ml tubes in the rotors
4. Pulse spin ALL tubes of aliquots to bring the liquid to the bottom of the tube -- in the microcentrifuge hold the "SHORT" key for about 5-10 seconds
5. DO NOT SLAM THE LIDS! (this action breaks the latch mechanisms)

Procedure for Pouring Plates

NOTE: do this procedure FIRST so the plates will ready for spreading after the transformation

Luria-Bertani (LB) plates, with the antibiotic ampicillin will be prepared today for plating transformations on day 3.  Each student will prepare 6 plates.

LB Agar:
Per liter:
1% Bacto-Tryptone = 10 g
0.5% Yeast extract = 5 g
NaCl = 5 g
Bacto Agar = 15 g
*adjust pH to 7.0 with NaOH and bring to 1 L; sterilize by autoclaving for 20 minutes at 121°C at 15 psi
**autoclaved media is always cooled to 50-60°C prior to the addition of antibiotics and some salts that are inactivated or precipitated by autoclaving.

Sterile technique
Media can be contaminated by contact with non-sterile surfaces or by air borne organisms. Remove lids and coverings carefully avoiding contact with any part of the cover that may contact the media. Lids and coverings should be held with media side down at all times. Air borne contaminants are usually falling downward. Replace the coverings carefully so that the rim of the container makes contact only with sterile surface of the inside of the cap.
The use of a flame helps maintain aseptic materials. Working near a flame can decrease air borne contamination. The flame is also used to singesurfaces to maintain sterility. The mouth of the tube or flasks is passed through the flame before and after pouring. The cap or cover is also passed through the flame prior to replacing on the container.

Caution: The flame is used to singe the surfaces only. Do not hold the items in the flame to make them hot. Glass flasks, even Pyrex, can break from the heat or when the cooler media hits the hot surface.

Label the bottoms of 6 Petri dishes. The bottoms are labeled because the lids can get separated. Also the plates are usually handled inverted. Some people label stacks of dishes with vertical marks on the edges of the lids. Different colors or different number of lines indicate the type of plate. If this method is used it is still advisable to mark the bottoms when the plates are used in case the lids are accidentally mixed.

1. Swirl large flask of LB-agar to mix contents well.
   The agar is held at 50-55°C in a water bath to prevent agar from congealing.
2. Transfer ~ 150 ml of LB-agar to a sterile flask.
   Use sterile technique - don't contaminate the media.
3. Using sterile technique, add ampicillin (Amp) [STOCK = 50 mg/ml in water (stored at - 20°C)] to the LB agar in a 1:1000 ratio (1 µl of antibiotic for every 1 ml of solution).
   Mix by swirling but avoid creating bubbles. Keep the solutions sterile.
4. Using sterile technique, pour 20-25 ml of LB-Amp agar into 10 Petri dishes; add agar solution until the bottom of the dish is covered.
   
   Work rapidly to pour the plates. The agar will begin to congeal in 5-10 minutes with the flask at room temperature.
   
   The plates can be placed individually on the surface of the desk or left in a stack while pouring. The stack method saves bench space and is accomplished by lifting the stack of 5 empty plates by the lid of the bottom most plate. Agar is poured into the Petri dish and the stack is replaced. Move your hand to the second lid and lift the stack again and pour the second dish of agar. Repeat until all the plates in the stack are filled.
   
   
5. Very carefully pass the flame over the surface of the agar to get rid of any bubbles.
6. Allow the plates to set at room temperature until they are congealed.
   This usually takes 30-45 minutes but check that the agar is set in the center plates by gently shaking.
7. Store the plates inverted at 4°C (sealed with plastic).

Bacterial Transformation: Electroporation of E. coli

1. Prechill 0.1 cm electroporation cuvette on ice
2. Thaw the electroporation-competent cells (TetS or TetR) on ice
   
   You must be extremely gentle when working with competent cells. These cells are highly sensitive to temperature changes and/or mechanical lysis. Mix cells by gently tapping the tube or swirling with a pipet tip, not by pipetting up & down or vortexing.
   
   
3. Add 1 µl of plasmid DNA to cells and gently mix; incubate on ice for 1 minute
   
   
   • BBa_I13521 (strong constitutive production of red fluorescent protein (RFP), in pSB1A2, AmpR)
   
   
4. Place the cuvette on its side and add cells + DNA (save the tube! put on a rack at room temp.)
5. Gently tap the cuvette until the mixture of cells and DNA settles evenly to the bottom (i.e., there is no gap across the cuvette)
6. Wipe outside of cuvette with KimWipe and slide the cuvette into the electroporation chamber until the cuvette connects with the electrical contacts
7. Pulse sample ONCE at 1.8 kV, 200 ohms, 25 µF (Bio-Rad Electroporator)
   **Record the time constant (in ms) and the actual volts (kV) delivered to sample**
   
   time constant (τ): the amount of time required for the actual voltage of the delivered pulse to decay to 1/e (37%) of the initial voltage
   {τ = Resistance (R) x Capacitance (C)}
   
   
8. QUICKLY remove the cuvette from the chamber and add 1 ml of prewarmed (40-44°C) sterile SOC medium to the cells
9. With a sterile Pasteur pipet, quickly but gently resuspend the cells and transfer the cell suspension to the original 2 ml tube
10. Incubate the sample at 37°C for 1 hour with shaking at 225 rpm
11. Pipet 200 µl of sterile water on a LB-ampicillin plate and add 1 µl of transformed cells to the pool of water
12. Pour 10 - 20 sterile solid glass beads onto the plate, set the plate on the benchtop, and "shake" plate in a perpendicular motion; invert plate to pour off beads (collect in a beaker -- these can be reused)
13. On a second LB-amp plate, pipet 200 µl of sterile water and add 1 µl of 1:1000 transformed cells (diluted in sterile water)
14. REPEAT step 12
15. Incubate the plates (upside down) overnight at 37°C
    
    ***You will need to come in Tuesday afternoon and pull 1 colony for an overnight culture***
    • Put 4 ml of LB-amp into a sterile 15 ml culture tube
    • Touch a sterile toothpick or a sterile pipet tip to a well-isolated colony then twirl the tip in the LB in one of the tubes (leave the toothpick or pipet tip in the tube)
      NOTE: a touch is sufficient -- Do not remove the entire colony or gouge the agar!
    • Place the tube, at an angle with lid slightly loosened, in the 37°C shaking floor model incubator
    • Store the plates at 4°C

Agarose Gel Electrophoresis

Gel Casting:

Agarose can become superheated and violently boil over. Exercise caution when heating. Swirl flask occasionally during heating. Heat until close inspection reveals that the agarose is 100% dissolved. Undissolved agarose will appear as little flecks that look like Lilliputian contact lenses.

Ethidium bromide is a powerful mutagen and is moderately toxic and should be handled with care. WEAR GLOVES when handling contaminated equipment or solutions containing ethidium bromide. Confine the compound to the restricted area. Use plastic wrap to protect equipment and surfaces from being contaminated.

Note: Concentrated ethidium bromide solutions should be decontaminated. One method is to treat 0.5 µg/ml staining solutions of EtBr with 1 g/liter activated charcoal, filter and incinerate the residue. Slurries of activated charcoal can be used to decontaminate surfaces (see Maniatis et al, (1989) for additional methods of decontamination).

Sample Preparation:

• Plasmid DNA:
  Concentration Standards -- Add 2 µl 6X LB to each plasmid standard (10 µl of 0.05, 0.1, and 0.2 µg/µl)
  Unknown -- Add 2 µl 6X LB to 10 µl plasmid DNA with "unknown" concentration
  
  
• Standards:
  Quick-Load® 1 kb DNA Ladder (New England Biolabs, Ipswich, MA): load 10 µl (0.5 µg) per lane
  
  
  
  *Record this information in your notebook and include both sizes (in bp) and mass of ladder fragments.
  **This ladder can be used to quantitate the amount of DNA in a sample; the mass of DNA in each band in the ladder has been calibrated (the 3,001 bp fragment has increased intensity to serve as a reference band).
  
  
  

  Gel Documentation with the UVP BioDoc-It™ System:

  System Components

  • CCD Video Camera
  • Zoom Lens
  • UV Blocking Filter--the orange-colored filter absorbs UV and IR radiation from the transilluminator and enhances the orange/pink bands of ethidium bromide stained gels
  • Transilluminator (302 nm)
  • Darkroom Cabinet
  • LCD Monitor
  • Thermal Printer

  Avoid doing anything that would intentionally contaminate the transilluminator or camera with EtBr. For instance, do not lay gels directly on the transilluminator, but always on plastic wrap. Do NOT contaminate the equipment (door knob, camera, printer, etc.)--REMOVE your gloves BEFORE working with the camera and gel documentation system.

  1. Position the gel in the center of the UV transluminator and smooth out the wrinkles in the plastic wrap.
  2. Close the darkroom cabinet's door and turn on the transilluminator.
     
     NOTE: the transilluminator will cut off if the darkroom door is opened.
     
     
  3. If the image on the LCD screen is dim, press the "+" button on the keypad controller; each time the "+" button is pressed, the CCD camera is set to the next increasing exposure time (there are 17 steps from 0.1 sec to 10 sec).
     
     NOTE: For more detail but a gray background, increase exposure time; for less detail and black background, decrease exposure time.
     
     
  4. Rotate the zoom adjustment (MIDDLE ring) on the lens so that the image is the appropriate size.
  5. Press the "Capture" button to hold the image on the screen.
  6. Press "Print" on the thermal printer in order to print the image; press AND hold copy/feed to advance the paper to cut the picture.

  Dispose of the gels in the Biohazard Waste Box.
  Do NOT put paper towels, plastic wrap, or gloves in this waste box--ONLY the gels.

  
  

  Estimation of DNA Amounts by Intensity of Ethidium Bromide Fluorescence

  If a DNA sample is too dilute to measure at 260 nm or is contaminated with other compounds that absorb in the UV range, the amount of DNA present can be estimated from the intensity of ethidium bromide fluorescence. Since the amount of DNA in a solution is proportional to the fluorescence emitted by ethidium bromide, the DNA quantity in an "unknown" solution can be estimated by comparing its level of fluorescence with the intensity of known amounts of DNA.
  
  
  • Compare the intensity of the "unknown" DNA to "known" plasmid standards
    1. Run an agarose gel comparing your "unknown" to "known" standards
    2. Photograph the gel
    3. Compare the relative intensity of staining of the unknown with the standards
    4. Estimate the DNA concentration of your sample
    
    
  • Also, compare the intensity (e.g., 2X as bright) of the "unknown" DNA to a band in the 1 kb ladder to estimate the DNA concentration of your sample (ng of DNA for each size standard are given in the Table above)
    How closely does this value agree with comparison with "known" plasmid standards?
  
  

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  Copyright, Acknowledgements, and Intended Use
  Created by B. Beason (bbeason@rice.edu), Rice University, 21 November 2007
  Updated 11 May 2009