Southern California Biotechnology Center (SCBC) at Miramar College: Amgen Biotech Experience (ABE) Program
Standard Operating Procedure (SOP): December, 2015
Title: Manually preparing LB, LB/AMP and LB/AMP/ARA Plates for Amgen Labs 5/5a/5b.
Equipment/Materials/Supplies:
Autoclave
Water bath
Stir plate
Electronic balance
Pipette aide
Serological Pipettes
Graduated cylinder (1 liter)
2 Liter Erlenmeyer flasks (3)
Spatula
Weigh boats
Stir bars
Sterile plates
Mitts
Autoclave tape
Aluminum foil
DI H2O
LB powder
Agar
Ampicillin
Arabinose
70% ethanol
Blue sharpie
Ampicillin:
Use Ampicillin powder or use Ampicillin stock which is 100mg/mL and is already prepared and stored at -20 °C
0.05 grams of ampicillin powder per liter LB agar. Use good sterile technique to weigh the ampicillin powder on a piece of weigh paper and dump it into the cool Lb Agar or if you have a 100mg/ml solution of ampicillin already prepared, use 1.0 mL ampicillin per liter LB agar (see step 12 below under the title: LB Agar + Ampicillin (LB/Amp) Plates
Arabinose:
2 grams of arabinose powder per liter LB agar. Use good sterile technique to weigh the arabinose powder on a piece of weigh paper and dump it into the cool Lb Agar (see step 12 below under the title: LB Agar + Ampicillin + Arabinose (LB/Amp/Ara) Plates
Luria Bertani (LB) Agar Plates:
Makes 1 L (about 35 - 40 plates). Store at 4°C for ~ 3 months.
1. Obtain a 2 L Erlenmeyer use a piece of tape and label the flask LB.
2. Gently place a stir bar into the flask.
3. Use a graduated cylinder and measure 1 L of dH20. Pour the water into the flask.
4. Use an electronic balance to weigh out 20.0 grams of LB and 15.0 grams of agar.
5. Add the 20 grams LB and 15 grams of agar to 1 L dH20 in the 2 L Erlenmeyer flask
6. Place flask on stir plate.
7. Stir to mix the LB and agar using a magnetic stir bar. The LB will go into solution but the Agar will not.
8. Cover the opening of the flask with aluminum foil and put a piece of autoclave tap on the foil.
9. Autoclave for 15 min at 121 °C and 15 PSI.
10. Set a water bath to 55°C
11. After autoclaving is complete use insulated gloves to carefully remove the solution from the autoclave. Use caution the solution will be very hot.
12. Place the solution into the 55°C water bath. Be careful do not let the flask tip over. Allow the temperature of the water bath to reach 55°C.
13. See the section below on how to sterilize your work area and how to pour plates.
14. Once plates are poured and cooled, mark the side of each lid with 1 blue line.
LB Agar + Ampicillin (LB/Amp) Plates:
Makes 1 L (about 35-40 plates). Store at 4°C for ~ 3 months.
1. Obtain a 2 L Erlenmeyer use a piece of tape and label the flask LB/AMP.
2. Gently place a stir bar into the flask.
3. Use a graduated cylinder and measure 1 L of dH20. Pour the water into the flask.
4. Use an electronic balance to weigh out 20.0 grams of LB and 15.0 grams of agar.
5. Add the 20 grams LB and 15 grams of agar to 1 L dH20 in the 2 L Erlenmeyer flask
6. Place flask on stir plate.
7. Stir to mix the LB and agar using a magnetic stir bar. The LB will go into solution but the Agar will not.
8. Cover the opening of the flask with aluminum foil and put a piece of autoclave tap on the foil.
9. Autoclave for 15 min at 121 °C and 15 PSI.
10. After autoclaving is complete use insulated gloves to carefully remove the solution from the autoclave. Use caution the solution will be very hot.
11. Place the solution into the 55°C water bath. Be careful do not let the flask tip over. Allow the temperature of the water bath to reach 55°C.
12. When the solution has cooled down to 55°C, place the solution on a stir plate and sterilely add 1.0 ml of ampicillin stock solution or 0.05 grams of ampicillin powder to the flask. Turn on stir plate and stir solution to ensure uniform mixing of the ampicillin throughout the solution.
13. Place the flask back into the 55°C water bath.
14. See section below on sterilizing your work area and pouring the plates.
15. Once plates are poured and cooled, mark the side of each lid with 2 blue lines.
.
LB Agar + Ampicillin + Arabinose (LB/Amp/Ara) Plates:
Makes 1 L (about 35-40 plates). Store at 4°C for ~ 3 months.
1. Obtain a 2 L Erlenmeyer use a piece of tape and label the flask LB/AMP/ARA.
2. Gently place a stir bar into the flask.
3. Use a graduated cylinder and measure 1 L of dH20. Pour the water into the flask.
4. Use an electronic balance to weigh out 20.0 grams of LB and 15.0 grams of agar.
5. Add the 20 grams LB and 15 grams of agar to 1 L dH20 in the 2 L Erlenmeyer flask
6. Place flask on stir plate.
7. Stir to mix the LB and agar using a magnetic stir bar. The LB will go into solution but the Agar will not.
8. Cover the opening of the flask with aluminum foil and put a piece of autoclave tap on the foil.
9. Autoclave for 15 min at 121 °C and 15 PSI.
10. After autoclaving is complete use insulated gloves to carefully remove the solution from the autoclave. Use caution the solution will be very hot.
11. Place the solution into the 55°C water bath. Be careful do not let the flask tip over. Allow the temperature of the water bath to reach 55°C.
12. When the solution has cooled down to 55°C, place the solution on a stir plate and sterilely add 1.0 ml of ampicillin stock solution or 0.05 grams of ampicillin powder to the flask and sterilely add 2 grams of arabinose to the flask. Turn on stir plate and stir solution to ensure uniform mixing of the ampicillin throughout the solution.
13. Place the flask back into the 55°C water bath.
14. See section below on sterilizing your work area and pouring the plates.
15. Once plates are poured and cooled, mark the side of each lid with 3 blue lines.
Sterilizing the work area before pouring plates:
1. Put on a pair of gloves. Prepare the plate pouring area by spraying the bench top with a generous amount of 70% ethanol or cavicide (disinfectant). Use paper towels to wipe down the work area and allow to air dry.
2. Obtain sterile Petri dishes. Spray and wipe the sleeves with paper towel and 70% ethanol (EtOH) before placing them in the work area.
3. Use scissors to cut open the top of the plastic sleeves. Do not destroy the sleeves they will be used to store the plates.
4. Remove the petri dishes from the sleeve and place them in the work area. Make sure the plates remain closed.
5. Make stacks of three to five petri dishes lid side up.
6. With one hand holding the neck of the flask, open the cover of the lowest Petri dish with the other hand, just wide enough to pour the sterilized agar.
7. Pour approximately 25 mL of agar into bottom plate. Cover the plate.
8. After pouring the bottom dish, pour the next higher plate, cover and repeat with the remaining plates.
Note: If this is your first time pouring plates, you can use a 25 ml sterile serological pipette to put the agar into the plates.
9. Once a stack of plates has been poured, slowly and carefully slide the stack to an unoccupied area of the bench where the plates will be left undisturbed for the agar to cool and solidify.
10. See step 15 above for labeling the plates once they have cooled.
Storage and Quality testing of Plates:
1. After plates have been labeled and have cooled completely, place them back into the sleeves and close with colored lab tape. Label the tape with the number of plates in the sleeve, type of plates, date poured, initials and “NEEDS QC”.
2. Store the plates agar side up at 4 °C.
3. Quality testing of the prepared plates must be performed before sending plates out to schools.
4. Test the plates by performing a transformation experiment. Amgen lab 5a.
5. After obtaining results of transformation experiment. Make the sleeve of plates as “Passed QC”, date tested and initials.