Maxiprep of KCNJ9_FLAG_pcDNA3.1

1. Go to the equipment room down the hall and lift the screen on the biological safety cabinet (a.k.a. the tissue culture hood). Find the control switches at the top. Flip the BLOWER switch to the ON position. Flip the STERILIZING switch to FLUORESCENT.

2. Spray the work area with ethanol and wipe with a paper towel.

3. Put gloves on and obtain a bottle of LB Broth+Amp (100 µg/mL, Fridge A) and add 75uL to each flask, the bacterial culture plates, a box of small pipet tips, and as many 250 mL Erlenmeyer flasks (Cabinet A) as needed. Label the Erlenmeyer flasks with the names of the DNA. Take all the materials to the biological safety cabinet.

4. Rub ethanol on your gloves and allow it to dry. Pour 75 mL of broth into each Erlenmeyer flask. Obtain a pipet tip, remove the lid on one of the plates and visually inspect for an acceptable colony. Good colonies will be medium size and separate from other colonies.

5. Once a colony is found, drag the pipet tip across the colony without touching other colonies. Ensure you have gotten as much of the colony as possible without getting another colony or scarring the agar. Drop the pipet tip straight down into its corresponding Erlenmeyer flask. Replace the lid on the plate and the foil on the flask.

6. After all flasks are inoculated, place them in the small incubator and shake at 37°C at 170 RPM for about 15-16 hours (until cloudy). Take all the materials back, clean the work area with ethanol, and lower the screen on the cabinet. Turn off the biological safety cabinet.

Choose a colony from the 1ng/uL plate

1. Go to the equipment room down the hall and lift the screen on the biological safety cabinet (a.k.a. the tissue culture hood). Find the control switches at the top. Flip the BLOWER switch to the ON position. Flip the STERILIZING switch to FLUORESCENT.

2. Spray the work area with ethanol and wipe with a paper towel.

3. Put gloves on and obtain a bottle of LB Broth+Amp (100 µg/mL, Fridge A) and add 75uL to each flask, the bacterial culture plates, a box of small pipet tips, and as many 250 mL Erlenmeyer flasks (Cabinet A) as needed. Label the Erlenmeyer flasks with the names of the DNA. Take all the materials to the biological safety cabinet.

4. Rub ethanol on your gloves and allow it to dry. Pour 75 mL of broth into each Erlenmeyer flask. Obtain a pipet tip, remove the lid on one of the plates and visually inspect for an acceptable colony. Good colonies will be medium size and separate from other colonies.

5. Once a colony is found, drag the pipet tip across the colony without touching other colonies. Ensure you have gotten as much of the colony as possible without getting another colony or scarring the agar. Drop the pipet tip straight down into its corresponding Erlenmeyer flask. Replace the lid on the plate and the foil on the flask.

6. After all flasks are inoculated, place them in the small incubator and shake at 37°C at 170 RPM for about 15-16 hours (until cloudy). Take all the materials back, clean the work area with ethanol, and lower the screen on the cabinet. Turn off the biological safety cabinet.

Chose a colony from the 10ng/uL plate.

1. Head to the large centrifuge room on the second floor with cart and bacterial cultures. Transfer 40 mL of the bacterial culture into a Falcon tube. Spin at 5,000 g for 3 minutes. Discard supernatant into waste jar. Repeat with the rest of the bacterial culture in the same tube.

2. Add about 15 mL of TENS buffer. Vortex for 10 seconds or until the pellet is dissolved.

3. Add about 7.5 mL of 3 M sodium acetate pH 5.2. Vortex for 10 seconds.

4. Centrifuge for 5 minutes at 10,000 g and transfer supernatant to a new, previously labeled Falcon tube.

Stopped in the middle of step 4 due to a centrifuge error.

1. Head to the large centrifuge room on the second floor with cart and bacterial cultures. Transfer 40 mL of the bacterial culture into a Falcon tube. Spin at 5,000 g for 3 minutes. Discard supernatant into waste jar. Repeat with the rest of the bacterial culture in the same tube.

2. Add about 15 mL of TENS buffer. Vortex for 10 seconds or until the pellet is dissolved.

3. Add about 7.5 mL of 3 M sodium acetate pH 5.2. Vortex for 10 seconds.

4. Centrifuge for 5 minutes at 10,000 g and transfer supernatant to a new, previously labeled Falcon tube.

Stopped in the middle of a step 4 due to a centrifuge error