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Position: Discussion forums > Bioscience biotechniques discussion forums > Proteomics and protein biochemistry forum
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Experiment Step Verification
Posted by: fr0gjaywalker (IP Hidden, New member, 3)
Date: July 8, 2005 05:16PM
I was hoping someone can help me verify whether the steps I'm taking to prepare my bacterial culture is correct. I'm slightly confused on the ampicillin step. Can someone also verfiy whether the amount I'm adding to the preculture is correct? The professor said the final concentration should be 0.1 grams ampicillin per Liter of medium.
1. Prepare 1 Liter of Luria broth in an aerated Erlenmeyer. This is the primary stock. 2. Transfer 100 mL from the 1 L into a smaller flask for the preculture. 3. Autoclave both flasks for 25 minutes. 4. Store the primary stock flask in the warm room (my professor said this is to prevent the bacteria from getting heat shock later when I dilute the preculture into the primary stock.) 5. Prepare and add the ampicillin to the preculture--for stock I made 0.1g/1.25 mL. Since the total FINAL concentration needs to be 0.1g/1000 mL, I added .125 mL to the preculture flask which had 100 mL of medium. 6. Inoculate the preculture. Put flask in warm room and shake. Bacterial growth overnight. Next day... 7. Add the ampicillin to the primary stock (I’m assuming I just add what’s left of my ampicillin stock?) 8. Pour preculture stock into primary stock. 9. Take spectrophotometer readings to determine bacterial growth density. 10. When bacterial growth is sufficient, pour into centrifuge tubes and centrifuge. Pour out LB, and add EDTA-Kphophate buffer (how much?) 11. Centrifuge again. 12. Bath cells in same buffer. 13. Add PMSF. Later I'll be putting the cells through the French press and do an assay with Rhodamine 6G using a machine called the spectrofluorophotometer. If anyone can describe to me how I operate the spectrofluophotometer (even though the professor will be helping me), I would greatly appreciate it! THANKS EVERYONE FOR YOUR HELP!
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