11 replies to this topic

[PhilS]

Hi,

I prepare my own sterile water for PCR reactions. Every few months. I autoclave ~50 x 1.5 mL eppendorf tubes, each with 1 mL of water inside. The tubes are clean and the water has come from a filtered water source in our lab. When I autoclave, I cover the tops of the tubes to prevent them bursting open. The eppendorf's are then stored in a screw top jar on my bench.

However, every time, after a month or so, I start to see small flecks of white appear in the water. Before I ignored it, knowing that they were still sterile, but as I've been having trouble with contamination recently, I started to suspect this might be the source. But when I look at these specs under the scope, I do not see any cells. I'm thinking of calling the manufacturer of the tubes to ask them for their advice, but I thought I'd post here first.

Any thoughts?

Cheers,
Phil

[sanjiun]

autoclave in an eppendorf tube...
hmm... i am not sure about other people. But i don't trust the "tightness" of the eppendorf cap especially when pressure is applied during autpclave.
Normally I would prefer autoclave in a glass bottle and aliquot into small eppendorf tubes in a hood.

[phage434]

I wouldn't autoclave water for pcr at all. Have you ever looked inside your autoclave? And you want that stuff in your reaction?

[sanjiun]

phage434, on Nov 17 2009, 10:48 AM, said:

I wouldn't autoclave water for pcr at all. Have you ever looked inside your autoclave? And you want that stuff in your reaction?

to be safe, you can buy the nuclease free water.
If your lab has a good duty rota and every lab member take turn to wash/ clean autoclave every week, OR, you have a separate autoclave for clean/ dirty stuff... then shouldn't be any problem.
My lab always use autoclaved water for PCR etc... it works fine.

[Vini]

in our lab, we use autoclaved water only.........what i do is, autoclave it in schott duran bottle..........n then make aliquots of it in eppendorf tubes.....anyhow, i don't use it for as long as 1 month. rather keep autoclaving at an interval of ~ 2weeks.

[Prep!]

even i ve always used autoclaved water always... and have had no probs.. (we do it the same way DRN said)
still you can use type I autoclaved too.. or WFI!!!

[gogreen]

white flakes in water can be salts which appear to be on the surface after boiling, Can u just boil the same water in a beaker and allow it to cool and check if it appears over the surface...If so, I would get my filter checked first!!

[PhilS]

Thanks all for notes, I think autoclaving in glass and pipetting into tubes in hood is my best bet.

gogreen, on Nov 17 2009, 12:06 AM, said:

white flakes in water can be salts which appear to be on the surface after boiling, Can u just boil the same water in a beaker and allow it to cool and check if it appears over the surface...If so, I would get my filter checked first!!

@gogreen, so you are saying it could be salts forming, (I think this is most likely) and if aI boil it then it should go away, otherwise it is something to worry about? Can you explain why would salts appear on the surface after boiling though?


Phil

[gogreen]

Sorry Phil, I am not good at the chemistry side of it..I saw the same thing happening once when I boiled the tap water at home...It was not immediately visible, I saw it the next morning..obviously no contamination in water Did u try boiling the water?? that would give you a first hand experience if this is same as what I suspect!!

[hobglobin]

should be then calcium/magnesium-carbonate...thought he'd have used deionised water (or even double deionised) as one should use (and then it's impossible that the white flecks are salts)... but don't know what "filtered water source" means...

Edited by hobglobin, 17 November 2009 - 11:46 AM.

[Feelcontraire]

PhilS, on Nov 16 2009, 05:22 PM, said:

Hi,

I prepare my own sterile water for PCR reactions. Every few months. I autoclave ~50 x 1.5 mL eppendorf tubes, each with 1 mL of water inside. The tubes are clean and the water has come from a filtered water source in our lab. When I autoclave, I cover the tops of the tubes to prevent them bursting open. The eppendorf's are then stored in a screw top jar on my bench.

However, every time, after a month or so, I start to see small flecks of white appear in the water. Before I ignored it, knowing that they were still sterile, but as I've been having trouble with contamination recently, I started to suspect this might be the source. But when I look at these specs under the scope, I do not see any cells. I'm thinking of calling the manufacturer of the tubes to ask them for their advice, but I thought I'd post here first.

Any thoughts?

Cheers,
Phil

Well, if it is salts then it should redisolve by shaking and/or heating.

If you put thimerosal or sodium azide at 0,1% in one of your eppendorfs and the flecks son't appear then it´s contamination.

If it is contamination you can just freeze the tubes and and place them at 4ºC just a few days before use to avoid the flecks growing without having to include extra-something to the buffer

[phage434]

If you water is any good, it will have essentially no salts of any kind, and will evaporate without any sort of residue. It also won't grow much (but will perhaps grow a very few bacteria, amazingly enough). Certainly not enough to actually see or to find on a microscope slide. I would worry about how pure my water was if I saw anything of any kind in it. What's the resistivity?