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Preventing contamination in TBS/T (WB wash buffer)


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5 replies to this topic

#1 Trof

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Posted 07 December 2018 - 03:04 AM

Hi,
we make our TBS/T stock like this:

50 ml 2M Tris pH7.6

70 g NaCl

8 ml Tween 20

dH2O up to 1 L

 

Now, twice already our stock wash got full of hairy mould. The first time the contamination was in the stock Tris as well, in some other buffers so I autoclaved all the bottles used for the next time.
But yesterday again, moulds. No other solution was afected this time. I don't think the stock Wash was opened more than a few times and expect normal circumstances.

 

I don't know what to do to prevent this, we don't do WB so often, so the stock may stay there for month or so. It is not very efficient get contamination again and again and remake it everytime before doing WB.

I wonder what can be improved. I'm not sure at all about the quality of our distilled water, but in other stocks it doesn't make such problems. I can have autoclaved dH2O. But what other options, can I autoclave TBS/T? Or just TBS and then add Tween later?

 

Thanks.


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#2 Curtis

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Posted 10 December 2018 - 06:29 AM

Can you keep it in a fridge?

#3 merlav

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Posted 10 December 2018 - 10:11 AM

I always use autoclaved bottles and water. Maybe the contamination comes from water. Make sure that the water dispenser is clean.  I clean the spigot with 10% clorox and a swab.  


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#4 OldCloner

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Posted 13 December 2018 - 08:57 AM

Do multiple users open the bottles?  I'd suggest aliquotting sterile stocks into smaller sterile containers in useful volumes- then open one aliquot once and use it all. or discard the leftover.



#5 mdfenko

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Posted 13 December 2018 - 11:31 AM

i always prepared my wb wash from stocks on the day that i used it. the stocks (tris and nacl concentrates) are stored in the fridge.


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#6 Trof

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Posted 02 January 2019 - 01:18 AM

Thank yoou all.

 

I found another option, make stock with everything except Tween and autoclave it. And add Tween before making 1x.

 

We do not store the stocks in a fridge. We have kind of limited fridge space, most of it take the bottles with transfer.

 

The water probably is the source though, I have no control about the dispenser, it is centralized. Not sure who takes care of it, if anyone. But I can autoclave the water, and see.


Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon





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