I first tried with 75 ug and i got cells growing but even the cells to which pUC19 was not added to grew in the media and so i increased the conc. to 150 ug and still the control grew in ampicillin plates and so i want to know how much ampicillin should be added to LB agar medium.
Usually concentration 50-100 microgram per ml of the medium is used. However, sometimes we increase it up to 200-250 microgram per ml. Do you use fresh ampicillin stock? How do you prepare and store ampicillin solution? Do you chill LB agar enough before the addition of ampicillin? I prepare fresh 100 mg/ml ampicillin stock in 50% ethanol and store it at -20 no more than for a week. Using HPLC we found, that ampicillin is unstable during long storage, so it's better to use fresh solution each time. Even ampicillin from one manufacturer, but with different lot numbers could work slilghtly different. If you've got problems with satellite growth of untransformed cells, and your ampicillin is ok, I recommend you to prepare LB agar plates with different amp concentrations and determine which one works better for your strain.
Usually concentration 50-100 microgram per ml of the medium is used. However, sometimes we increase it up to 200-250 microgram per ml. Do you use fresh ampicillin stock? How do you prepare and store ampicillin solution? Do you chill LB agar enough before the addition of ampicillin? I prepare fresh 100 mg/ml ampicillin stock in 50% ethanol and store it at -20 no more than for a week. Using HPLC we found, that ampicillin is unstable during long storage, so it's better to use fresh solution each time. Even ampicillin from one manufacturer, but with different lot numbers could work slilghtly different. If you've got problems with satellite growth of untransformed cells, and your ampicillin is ok, I recommend you to prepare LB agar plates with different amp concentrations and determine which one works better for your strain.
Thank you so much for your valuable suggestion Anna , i shall try it out and let u know you .
You are welcome! Hope, that it will help you with your experimental progress!
Best wishes!
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