how to work out how much IPTG/X-gal for plating

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biology_06er
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how to work out how much IPTG/X-gal for plating

Post by biology_06er » Fri Aug 05, 2011 11:55 am

Hi there,

The other day I was preparing agar plates for the first time and today I had to redo it using a slightly different method regarding adding the IPTG/X-gal.

The first time I was told to separately add 5uL of IPTG and 50uL of X-gal to each plate and spread it but the second time to make it a bit easier I was told to add IPTG/X-gal to the bottle of LB-agar before pouring. I had 350mL of LB-agar and to it I added (Well was told to add) 70uL IPTG and 700uL. Can anyone tell me how she would have worked this out...I can't recall the conc. of IPTG/X-gal but if someone can help me with the general calculation she would have used..would be greeeatly appreciated.

I used 50uL of omnimax cells and 5uL of my DNA sample if that helps

Thanks,
b_06er

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canalon
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Post by canalon » Fri Aug 05, 2011 12:33 pm

It is all to do with C1xV1=C2xV2, you should know your starting concentrations and the final concentration that you aiming for. After it is just a question of proportion, if your volume is N times larger than the plate, just add N times more of the reagent.
So all you need to know is either the volume of your plate or of your LBA bottle.
Keep in mind that in that case even if your concentrations are a little off, it is not a massive problem, as long as you have enough to induce and produce a color change. good concentrations are slightly more important with antibiotics as with too little, you will have background, and with too much and you have nothing.
Patrick

Science has proof without any certainty. Creationists have certainty without
any proof. (Ashley Montague)

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