Diluting Ampicillin into LB Agar - (Mar/21/2009 )

Hi
Thanks for ur explanation. I kept myself cool and worked out....I could get the concentration and the loading amount too.

I appreciate the clear explanation of this.

I feel like the answer is staring me in the face and I'm still having trouble figuring this one out - maybe you guys can help me.

I have a 5mg/mL LPS concentration in 20 ul

I have 200ul of DC cells in my well (2 X 10^5 cells total in each well)

I want to make a working stock of 50ug/mL LPS and transfer 10ul into my wells.

From there I want to make 1:5 dilutions of LPS (50, 10, 2, 0.4)

How do I start to make my LPS stock?
Please explain in detail as I have the calculation but I want to figure this out completely.
Thanks.

Please read the rest of this thread - it will answer your questions with a bit of thought. Bob

Hey guys,

I'm very new to the lab, but I'm starting to learn alot.

Just doing some routine cloning, I'm fine with everything except after weeks, I still haven't figured out how to dilute my ampicillin stocks! This is both embarrassing and ridiculous so I'm asking for some help if anyone can teach me on hwo to do it and I can apply this to everything else with regards to dilutions. I have no problems diluting 50X TAE to 1X TAE but I don't know why with ampicillin it is such a huge problem ):

Anyway, so I have made up 100mg/ml of Ampicillin stocks from powder.

I want to make up LB agar plates using 30mls of liquified LB agar.

How much ampicillin should I add if i want a concentration of 100ul/ml?

I know my stocks is at 1000X how do I go from here? Should I duilte to make working stocks of 1 or 10X first?

I am making 10 plates so that makes it to 300mls of LB Agar but I usually aliquot the ampicillin separately into plates that have been poured out with 30mls of LB agar Already (still in liquid form)


Appreciate all help! thanks a million!
Presuming you want ug/ml not ul/ml use the 1:1000 dilution. Keep your units the same for all variables. Bob.

It's a bad idea to add the ampicillin to the plate. You'll never be able to mix it properly. You have a 1000x solution, and you want to make a 1x solution, so .001 of your final volume should be ampicillin. If you are making 300 ml, you need 0.3 ml of ampicillin (and if you are being fussy, 299.7 ml of your agar, but no one does that). Make sure that the agar is cooled to about 55 before adding the ampicillin. The easy way to check is to see if you can pick the bottle up without burning your fingers.