Autoclaving nuclei isolation buffer - (Oct/16/2008 )

I have a quick general question, say I need to perform nuclei isolation. Do you think it's necessary to autoclave my lysis buffer? I add sucrose to it so I think it might not be sterile, other than that, all the other reagents are autoclaved.

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I would rather filter the buffer. But in my opinion, you don't need sterile buffers.

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Thanks for the reply Madrius, I guess for the protocol, a lot of these compounds are just for gradient reasons, so yea nothing should grow in it i guess. But do you think I should try and make my stock buffer solution sterile, for example, HEPES or PIPEs?

Also to since I'm quite new at using nucleus extraction buffers, does anyone know if PIPES can be autoclaved? I found that most ppl don't autoclave HEPES, eventhough some do and reported that it was no problem. I autoclaved mine for 30 minutes, now I'm having a little doubt after reading about HEPES.

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why not make a sucrose solution first, autoclave and then add to the rest of the gradient reagents?

sometimes filtering would change the composition and molarity.

if you have protease inhibitor then filtering or autoclaving is out of the question.

I never filter or autoclave my lysis buffers. because the amount of Tris, CHAPS, HEPES, Nacl, NP40, Triton, Na.Deoxycholate, EDTA etc. is so low that I'm scared to do that.

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I thought about making sucrose buffer, but I was told by someone in the lab that autoclaving sucrose turns some into glucose, wouldn't that affect the osmolarity as well? However, I guess if I make like a 5M sucrose stock without sterilizing, nothing should grow in it. I will do that next week.


And yea I add protease inhibitor right before I use them. Btw, how did you make your HEPES stock solution? did you autoclave them? I'm really interested to know because I autoclaved mine for 30 minutes and was wondering if I should toss them out and do it again. Also did you make stock Triton or Na.Deoxycholate solution or added them as you make the working buffer?

Thanks a lot

Marc

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I have a commercial RIPA from Pierce now, but I used to make it by myself. never made stock solution or autoclave...I used them right away, and trust me my RIPA worked even better than Pierce's.

why are you so concerned about autoclaving HEPES anyway?

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You never made HEPES stock? How do you use them right away, wouldn't that mean you have to pH them every time? Sry if these question seem newbish, I"ve done a nuclei isolation before but that buffer used Tric-Cl which we already have a stock of.

We have HEPES powders in a bottle, I assumed you'd have to create a stock solution from that no?

Marc

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I weigh HEPES just before use.

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So say if the protocol calls for HEPES pH 7.5, you add your HEPES and then pH it? And do you also weigh and add your protease inhibitor before use?

Thanks

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