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Diluting Ampicillin into LB Agar

LPS Flow Cytometry

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24 replies to this topic

#16 Golaso

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Posted 20 May 2011 - 02:47 PM

Hi all,

I'm new to this website and I found it helpful in reading some of the interested topics

just to mention that I started my undergrad research in purifying RNA mainly H69,

I really need some help in some of the calculations concerning O.D readings

So basically I ran a 1 mL reaction for 4-5 hours, centrifuge to remove any salts, do ethanol precipitation and disolve the pellet in dH2O.

then I take the O.D of the sample and I read it as let say it's 100 O.D at 260nm witha volume of 70 uL
now if I want to make the reading to be 20 OD (since i need to run it in a small 9 by 7 gel) how much of the 100 O.D sample should be taking and how much should i dilute it to.

If there is a website that you think would be helpfull to me to look at, please post it

I will be posting more question, cz I really need help with this as an undergrad

Thanks
Golaso

#17 pito

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Posted 25 May 2011 - 01:27 AM

If you need 20 OD, you simple need to dilute the 100 OD 5 times...

Its best to make a new post with questions like this.

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#18 tical84811

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Posted 10 December 2011 - 07:00 PM

Wish i found this website 1 year ago when i first started in the lab. However i've found it now and so far the content is amazing!

#19 Divya Pothula

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Posted 12 February 2012 - 09:56 PM

Hello Everyone!

I am kinda confused with the dilutions.....
1.I need to make a 0.02% of Bromophenol blue (working solution) in a 5Xor 10X buffer. I made 5X buffer and then made a 2% firstly.
by adding 0.16 gm to 6.4ml 5X buffer (made 8ml).

Now I need to make a 0.02 or0.01 final dilution. How do i do this?

2. I know the final concentration of my RNA extracted. I need to know how much I am loading or using /ul. I get so annoyed calculating in terms of ug/ul.

Can someone help me please.

Thanks

#20 mdfenko

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Posted 14 February 2012 - 08:23 AM

c1v1=c2v2

concentration, in your case, is weight per volume. g/l=mg/ml=ug/ul. if you know the concentration then you know how much you are loading/ul.
talent does what it can
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#21 Divya Pothula

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Posted 20 February 2012 - 05:01 PM

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

#22 jeanbaker

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Posted 10 May 2012 - 09:20 AM

I appreciate the clear explanation of this.

#23 watsonkatherine

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Posted 25 September 2012 - 03:56 PM

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

#24 izt_is

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Posted 28 September 2012 - 08:55 AM

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.

#25 phage434

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Posted 28 September 2012 - 08:53 PM

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.




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