- Sterilize lab bench with 70% ethanol.
- Remove overnight culture from the incubator.
- Remove the lid of the culture using sterile technique.
- Transfer all of the culture from one flask into one empty 500 ml centrifuge bottle. Repeat with the rest of the cultures.
- Make sure that the bottles are balanced.
- Place the A-4-81 roter in the centrifuge.
- Centrifuge at 4000 g for 20 minutes.
- Once the centrifuge is done, make sure that the cells are pelleted at the bottom of the bucket.
- Carefully pour the supernatant into a waste beaker.
- Add bleach to the waste beaker to a final concentration of 10%.
- Label a 50 ml centrifuge tube with the name of the bacteria, the plasmid, the date, and your initials.
- Add 5-10 ml of 1X lysis buffer to each bucket.
- Using a spatula, scrape all of the cells off the bottom of the buckets.
- Pour the cells out of the buckets and into the 50 ml centrifuge tube.
- Fill another 50 ml tube with water until it is the same weight as the first tube. This will act as a balance in the centrifuge.
- Replace the A-4-81 rotor with the A-4-62 centrifuge rotor.
- Centrifuge the 50 ml test tubes at 4000 g for 40 mins.
- Discard the supernatant.
- Store pelleted cells in freezer.
- Sterilize culture flask with bleach.
Note: When pelleting multiple cultures, try to centrifuge as many cultures as possible per cycle to save time. Label each test tube with the culture number to keep track of which culture goes in which tube.
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