I'm currently in a project involving CRISPR Cas9 for gene knockout and have initiated gene cloning following the protocol from the Zhang lab. My next steps include performing a ligation reaction followed by bacterial transformation. According to the Addgene website, it is recommended to use Ampicillin at a concentration of 100 μg/mL to promote bacterial growth. As such, I need to prepare 125 mL of agar plates. However, I'm unsure how to calculate the required amount of Ampicillin. I have Ampicillin that has been pre-diluted to a 1000x concentration in a 1.5 mL tube, but this solution was prepared by someone else. The original Ampicillin stock is 5g, with a molecular weight of 371.39 g/mol. I was considering using the equation C1V1=C2V2 to calculate this, but I'm missing the initial concentration since I wasn't the one who prepared the 1000x Ampicillin solution. This is my first experience working with bacteria, and I find it somewhat confusing. I would greatly appreciate any assistance you can provide.
It is a 100mg/ml stock (1000x) This means you just need 1μl per ml of media. Technically ampicillin selection still works with only 50μg/mL but the higher concentration is used for more stringency.
please , Would you mind explaining how to calculate this?
and thanks a lot for your respons!
if the stock solution is 1000X then you just calculate volume per volume. For 1 liter of media you would use 1ml of the stock. For 500 ml of media you would use 0.5 ml of stock. So in other words 1/1000 of the volume of media you have.
- Badges
- Science topic
- Similar topics
- Philosophy
- Philosophy Of Science
- Reasoning