Procedures

1. Safety Precautions
A. Basic Precautions:
1.Access to the laboratory is limited or restricted at the discretion of the laboratory director when experiments are in progress.
2. Persons wash their hands after they handle viable materials, after removing gloves, and before leaving the laboratory.
3. Eating, drinking, smoking, handling contact lenses, and applying cosmetics are not permitted in the work areas. Food is stored outside the work area in cabinets or refrigerators designated for this purpose only.
4. Mouth pipetting is prohibited; mechanical pipetting devices are used.
5. Access to the laboratory is limited or restricted by the laboratory director when work with infectious agents is in progress.
6. The laboratory director establishes policies and procedures whereby only persons who have been advised of the potential hazards and meet specific entry requirements (e.g., immunization) may enter the laboratory.
7. A biohazard sign is posted on the entrance to the laboratory
8. Biosafety procedures are incorporated into standard operating procedures or in a Biosafety manual adopted or prepared specifically for the laboratory by the laboratory director. (See aseptic techniques below)
9. The laboratory director ensures that laboratory and support personnel receive appropriate training on the potential hazards associated with the work involved.
10. A high degree of precaution must always be taken with any contaminated sharp items, including needles and syringes, slides, pipettes, capillary tubes, and scalpels.
a. Needles and syringes or other sharp instruments should be restricted in the laboratory for use only when there is no alternative.
b. Only needle-locking syringes or disposable syringe-needle units (i.e., needle is integral to the syringe) are used for injection or aspiration of infectious materials.
c. Syringes which re-sheathe the needle, needle-less systems, and other safety devices are used when appropriate. Containers of contaminated needles, sharp equipment, and broken glass are decontaminated before disposal, according to any local, state, or federal regulations.
d. Broken glassware must not be handled directly by hand, but must be removed by mechanical means such as a brush and dustpan, tongs, or forceps.
12. Laboratory equipment and work surfaces should be decontaminated with an effective disinfectant on a routine basis (10% bleach solution will be used).
13. Spills and accidents are immediately reported to the laboratory director.
B. Safety Equipment:
1. Properly maintained biological safety cabinets is available. PBHS has the Air flow 6000 which is a Class II Biosafety cabinet. This cabinet includes, has a UV light with timer and alarm. Close-able Plexiglas front entrance with a vertical flow Hepa filter.
2. PBHS also has readily available an autoclave which will reach maintain a pressure of 15 psi and a temperature of 120 degrees Celsius.
3. Face protection (goggles, mask, face shield or other splatter guard) is used for anticipated splashes or sprays.
3. Protective laboratory coats, gowns, smocks, are worn while in the laboratory. This protective clothing is removed and left in the laboratory All protective clothing is either disposed of in the laboratory or laundered by the institution; it should never be taken home by personnel.
4. Gloves are worn when hands may contact potentially infectious materials, contaminated surfaces or equipment.
C. Laboratory Facilities (Secondary Barriers):
1. Area has lockable doors for facilities that house restricted agents
2. Laboratory is away from public areas.
3. Each laboratory contains a sink for hand washing.
4. No Carpets and rugs are located in the laboratory.
5. Bench tops are impervious to water and are resistant to moderate heat and the organic solvents, acids, alkalis, and chemicals used to decontaminate the work surfaces and equipment.
6. An eyewash station and shower is readily available.
D. Aseptic Techniques:
1. Upon entering the lab, wash hands and arms up to the elbow with antibacterial soap. 2. Before and during experimentation wear rubber gloves, apron, and goggles at all times.
3. Use 10 % bleach solution and wipe down the lab area and tabletop.
4. The Transfer of any culture will be done with a mechanical pipette.
5. Place a biohazard sign in plain view for all to see near or at the testing site.
6. Loops and Needles used to transfer the culture will be sterilized by flame heating until bright red prior to use.
7. Needles will be capped at all times.
8. After experimentation, lab counter is cleaned with 10% bleach.
9 Upon leaving the lab, wash hands with 10% bleach.
Disposal: Petri dishes and other disposable equipment will be placed in a plastic bag and autoclaved for 30 minutes at 20 psi, 120 degrees Celsius and disposed of. All related glassware and related equipment would be autoclaved for 30 minutes at 20 psi, 120 degrees Celsius.
E. Autoclave:
1. Always wear goggles and aprons when using the autoclave.
2. The bottom of the auto clave should have about 2 inches of cleaned distilled water before operation.
3. Check the pressure release value prior to use (it should be clear)
4. Use potholders when opening or handling hot surfaces.
5. Opening the autoclave can be done only after the sterilizer after it has cooled (gauge should read zero.) and the all steam has been allowed to escape.
6. Place the control knob in the straight up position. This allows the unit to operate at 16-21 psi range.
F. UVA, UVB, and UVC light safety:
1. Limit UV exposure by placing the light source in a solid sealed case.
2. Wear UV protect goggles.
3. Wear personal protection includes aprons and gloves.
4. Never look directly into UV light source.
5. When using UVC, the light and reflection of light must be completely blocked before the unit is turned on.
6. Place a warning sign in the lab area that a UV light source is being used.

G. Radiation Safety:
1. Wear goggles or face shield when dealing with radiation sources.
2. Wear long sleeve shirts and pants.
3. Wear gloves and lab coats when handling and working around radiation sources.
4. Place a warning sign indicating low level radiation is being used in the lab area.
5. Radiation source must stored and locked when not in use.
6. Eating, drinking, and application of cosmetics in the laboratory is not permitted
7. Before leaving the lab, wash your hands thoroughly.
8. Keep exposure time to a minimum.
2. Set-Up Procedures
A. Approximate Time Requirements:
1. This experiment requires the reconstitution and incubation of Lyphocells at 34⁰-37⁰C for 16-24 hours before testing (over night incubation). Plan accordingly. For optimal results, incubate Lyphocells at 34⁰-37⁰C for 19 hours.
2. The Agar plates can be prepared several days in advance and stored inverted (agar side on top) in the refrigerator. Preparation requires approximately 1 hour.
3. Dispensing the DNA and control buffer requires approximately 30 minutes. This can be done the day before the lab and stored in the refrigerator.
4. Competent cells must be dispensed just prior to the lab experiment. If tubes are already labeled, dispensing will require approximately 15 minutes.
5. Allow ample time for the equilibration of the water baths at 37⁰C and 42⁰C and a bacterial oven at 37⁰C on the day of testing.
6. Testing will take approximately 50 minutes to plate a group of 3 bacterial cells.
7. Overnight incubation of plates is approximately 15-20 hours at 37⁰. Colonies will also appear between 24-48 hours at room temperature.
8. Follow disposal procedures as outlined in the section regarding Laboratory Safety.
B. Pour Agar Plates:
• For optimal results, prepare plates two days prior to plating and set aside the plates inverted at room temperature.
• If they are poured more than two days before use, they should be stored inverted in the refrigerator. Remove the plates from the refrigerator and store inverted for two days at room temperature before use.
1. Equilibrate a water bath at 60⁰C for step 5 below.
2. Loosen, but do not remove, the cap of the ReadyPour medium bottle to allow for the venting of the steam during heating.
3. Squeeze and vigorously shake the plastic bottle to break up the solid agar into chunks.
4. Heat the bottle of the ReadyPour medium by one of the two methods below. When completely melted, the amber-colored solution should appear free of small particles.
A. Microwave method:
• Heat the bottle on High for two 30 second intervals.
• Using a hot glove, swirl and heat on High for an additional 25 seconds, or until all the medium is dissolved.
• Using a hot glove, occasionally swirl to expedite melting.
B. Hot plate or Burner Method
• Place the bottle in a beaker partially filled with water.
• Heat the beaker to boiling over a hot plate or burner.
• Using a hot glove, occasionally swirl to expedite melting.
5. Allow the melted ReadyPour medium to cool. Placing the bottle in a 60⁰C water bath will allow the agar to cool, while preventing it from prematurely solidifying. When the ReadyPour medium reaches approximately 60⁰C, the bottle will be warm to the touch but not burning hot.
6. Use lab marker to “stripe” the sides of twenty (20) 60x15 mm petri dishes. This will provide an easy method of differentiating between plates with ampicillin.
• Open one sleeve of 20 plates and stack the plates neatly.
• Start the marker at the bottom of the stack and move the marker vertically to the top plate to “stripe” the sides of the 20 plates.
• These plates will be used with ampicillin.
• DO NOT stripe the second sleeve of plates. These will be the control plates.
7. Thaw and add all of the X-Gal solution (Component E) to the molten and cooled ReadyPour medium. Recap bottle and swirl to mix.
8. Use fresh 10 ml pipet pump to pour 10 unstriped plates, 5 ml each. Save the pipet for step 11.
9. Add the ampicillin power (entire contents of tube D) to the remaining molten ReadyPour medium.
10. Recap the bottle; swirl to completely dissolve the ampicillin powder.
11. Use the pipet from step 8 to pour 20 striped plates, 5 ml each. Pour extra plates with any remaining medium.
12. Allow the agar to cool and resolidify.
Note: If plates are to be used within two days, store in a sealable plastic so the plates will not dry out. Store at room temperature, inverted.

C. Preparation of Competent Cells:
1. Day before testing. Use a 10 ml sterile pipet to add 6 ml sterile cell reconstitution medium (Component f) to the vial of LyphoCells.
2. Replace the rubber stopper and cap. Mix by inverting until the freeze dried plug is dissolved.
3. Shake the cell suspension vigorously and incubate the vial at 34-37⁰C for 16-24 hours (overnight) in an incubation oven. For optimal results, incubate LyphoCells for 19 hours.
4. Day of testing. Completely thaw the competency induction solution (G) and place on ice. (If there is a white precipitate in the bottle, warm it in a 37⁰C waterbath to dissolve the precipitate.)
5. Mix and resuspend the vial of incubated cells by inverting and gently shaking. Place the vial on ice for 10 minutes
6. Use a 10 ml sterile to add 3 ml of ice cold competency induction solvent (G) to the vial of cells. The competency of induction is very viscous. Make sure that a portion of the solvent is not left on the walls of the pipet.
7. Mix the cells and induction solvent thoroughly by inverting the vial several times. The solution should have no dense layers, “streams” or globules (i.e. it should be uniform suspension).
8. Keep the cells on ice for a minimum of 30 minutes. Cells can be kept on ice for up to 3 hours.
9. Dispensing cells just prior to testing. Mix the cells by inversion to obtain an even suspension.
10. Use a sterile 1 ml pipet to aliquot 0.7 ml of cells to 10 ice cold ice cubes labeled “Cells”.
11. Cap the tubes and keep them ice.

D. Preparation of DNA and Control Buffer:
1. Place the tubes of supercoiled pGAL DNA (Component B) and control buffer (Component C) on ice.
2. Before dispensing DNA and Control buffer, tap the tubes until all the sample is at the tapered bottom of the tube.
3. Using an automatic micropipet, dispense .25 ml of the supercoiled pGAL DNA to each of the 10 micro test tubes labeled “pGAL DNA”.
4. Cap the tubes and place them on ice.
5. Using a FRESH micropipet tip, dispense 25 µl of control buffer to each of 10 micro test tubes labeled “Control Buffer”.
6. Cap the tubes and place them on ice.

E. Be Careful to Avoid Common Pitfalls:
1. When heating the ReadyPour medium, make sure it does not boil over and cause the volume to be reduced. Watch the bottle very carefully and remove it from heat if you see signs of the medium boiling over.
2. If the plates are made fresh, the plated cells will take longer to be absorbed into the medium, Invert the plates only after the cell suspension has been absorbed.
3. Do not discard the plates containing transformed bacteria. After plating an aliquot on selection plates, set the tubes in a rack and leave on the lab bench overnight. If, for some reason, transformants do not grow on the selection plates the cell pellet can be plated as follows:
• Collect the bacterial cell pellet by centrifugation in a micro centrifuge. If a micro centrifuge is not available, let the bacteria collect by gravity and do not disturb.
• Remove all except 0.1 to 0.2 ml of the medium (supernatant).
• Resuspend the cell in the remaining medium.
• Spread entire contents of the tube on the selection medium.
• Incubate plate as before, 15 to 24 hours at 37⁰C.

F. Streaking Plates:
1. Sterilize work area and don safety equipment.
2. Take out plates from refrigerator and allow to warm up to room temperature.
3. Take out bacterial culture to be streaked onto plates.
4. Take inoculating loop and scrape off growth from bacterial culture.
5. Place inoculating loop into sterile broth of distilled water and agitate.
6. Dip sterile swab into wash.
7. Lift up lid of plate and streak wet swab onto plate being sure to cover the entire area. Plate can be streaked multiple times in a sweeping motion to ensure plate overage.
8. When done streaking plates, put into incubator upside down. Depending upon the bacteria, growth can appear as soon as 24 hours later.
9. Dispose of materials.

G. Cleaning up:
1. After each day of experimentation, clean the lab counter with 10% bleach.
2. Wash hands with 10% bleach.
3. Clean with 10% bleach all glassware and related items. Auto clave new equipment as needed.
4. Petri dishes and other disposable equipment will be placed in a plastic bag and autoclaved for 30 minutes at 20 psi, 120 degrees Celsius and disposed of.
5. All related glassware and related equipment would be autoclaved for 30 minutes at 20 psi, 120 degrees Celsius.

H. Disposal:
1. Items of biological hazard must be disposed of after autoclaving.
2. Put on protective equipment (goggles, gloves, aprons).
3. Gather biological items to be autoclaved prior to disposal.
4. Put items into autoclavable bag. Put bag in autoclave.
5. Autoclave for thirty minutes at 20psi, 120 degrees C.
6. When able, remove from autoclave and transport to dumpster.
3. Experimentation Procedures:
A. Determining UV Exposure Levels:
1. On the bottom of a Petri-dish mark out 10 columns and label from left to right 10 down to one (representing the levels of UV exposure).
2. Across the center and perpendicular to the columns, mark out a central row in which the E. coli will be swipped.
3. With an inoculating loop streak one line of E. coli in the central row.
4. Obtain a note-card large enough to cover the Petri-dish in the UV light.
5. Repeat this two more times, now there should be one Petri-dish prepared for UVA, UVB and UVC.

B. Setting up the Transformation and Control Experiment:
1. Put initials on tubes labeled “pGAL DNA” and “Control Buffer”.
2. For the control: Using a sterile pipet, transfer 0.25 ml (250µl) of cell suspension from the tube “Cells” to the tube “Control Buffer”. Carefully place the pipet back into the wrapper. Cap the tube; mix by tapping. Put the tube back on ice.
3. For the transformation: Using the same sterile pipet from Step 2, transfer 0.25 ml (250µl) of cell suspension from the tube “Cells” to the tube “pGAL DNA”. Cap the tube; mix by tapping. Put the tube back on ice.
4. Incubate the cells prepared in steps 1-3 on ice for 10 minutes.
5. Place both transformation tubes at 42⁰C for 90 seconds. This heat shock step facilitates the entry of DNA in bacterial cells.
6. Return both tubes to the ice bucket and incubate for 1 minute.
7. Add 0.75 ml of the Recovery Broth to the tube “Control Buffer”. Add the recovery broth with a sterile 1 ml pipet. Avoid touching the cells with the pipet.
8. Add 0.75 ml of the Recovery Broth to the tube “pGAL DNA”.
9. Incubate the closed tubes in a 37⁰C water bath for 30 minutes for a recovery period.
10. After the recovery period remove the tubes from the bath and proceed to the UV radiation phase.

C. Plating the Cells:
1. Use a fresh, sterile 1 ml pipet to transfer recovered cells from the tube “Control Buffer” to the middle of the following plates: 0.25 ml to the plate labeled XGAL/Control 1 and 0.25 ml to the plate labeled AMP/XGAL/Control 2.
2. Spread the cells over the entire plate with a sterile inoculating loop.
3. Cover broth control plates and allow the liquid to be absorbed. Do not set the lid down on the lab bench, lift the lid enough only to perform inoculation.
4. Use a fresh, sterile 1ml pipet to transfer recovered cells from the tube “pGAL DNA” to the middle of the following plate: 0.25 ml to the plate labeled AMP/XGAL/pGAL.
5. Spread the cells with a sterile inoculating loop.
6. Covert the plate and allow the liquid to be absorbed (approximately 15-20).
7. Group similar plates together and tape off, label these groups properly. Plates should be left in the upright position to allow the cell suspension to be absorbed by the agar.
8. Place the plates in a common and safe place.
9. After the cell suspension is absorbed by the agar, the plates will be inverted (agar side on top)in a 37⁰C bacterial incubation oven for overnight incubation (15-20 hours). If the cells have not been absorbed into the medium, it is best to incubate the plates upright. The plates are inverted to prevent condensation on the lid, which could drip onto the culture and may interfere with experimental results.
10. Count the number of colonies on the plate and count the number transformed.